Chlamydia PMP Proteins, Gene Sequences and Uses Thereof

ABSTRACT

The invention discloses the  Chlamydia  PMPE and PMPI polypeptide, polypeptides derived therefor, (PMP-derived polypeptides), nucleotide sequences encoding said polypeptides, antibodies that specifically bind the PMP polypeptides and PMP-derived polypeptides and T-cells specific for PMP polypeptides and PMP-derived polypeptides. Also disclosed are prophylactic and therapeutic compositions, including immunogenic compositions, e.g., vaccines, comprising PMP polypeptides or PMP-derived polypeptides or antibodies thereto. The invention additionally discloses methods of inducing in animals an immune response to  Chlamydia  cells,  Chlamydia  elementary bodies, and/or cells expressing Chlamydial proteins, e.g., cell infected with  Chlamydia.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.09/677,752, filed Oct. 2, 2000, which is hereby incorporated byreference in its entirety.

1. FIELD OF THE INVENTION

The present invention generally relates to polymorphic membrane proteinsor PMPs of Chlamydia, amino acid and nucleotide sequences thereof,antibodies specific for such Chlamydia PMP polypeptides, prophylacticand therapeutic compositions, including vaccines, and to methods ofpreventing, treating or ameliorating disorders in mammals and birdsrelated to Chlamydia infections and for inducing immune responses inanimals to Chlamydia.

2. BACKGROUND OF THE INVENTION

Chlamydiae are obligate intracellular bacteria that infect animals,including mammals and birds, particularly at the epithelial lining ofthe lung, conjunctivae or genital tract. The most common species ofChlamydia include Chlamydia trachomatis, Chlamydia psittaci, Chlamydiapecorum and Chlamydia pneumoniae. Recently, the newly designated speciesof Chlamydia, C. pneumoniae (formerly C. trachomatis TWAR), has beenimplicated as a major cause of epidemic human pneumonitis and perhapsmay play a role in atherosclerosis.

There are currently 18 recognized C. trachomatis serovars, causingtrachoma and a broad spectrum of sexually transmitted diseases, with theA, B and C serovars being most frequently associated with trachoma,while the D-K serovars are the most common cause of genital infections.

Chlamydia are prevalent human pathogens causing disorders such assexually transmitted diseases, respiratory diseases, includingpneumonia, neonatal conjunctivitis, and blindness. Reactive inflammatoryarthritis is a common sequel to sexually acquired non-gonococcal genitaltract infection. Approximately 50% of reactive inflammatory arthritiscases are associated with Chlamydia trachomatis infection of the genitaltract.

C. trachomatis is the major cause of sexually transmitted disease inmany industrialized countries, including the United States. While theexact incidence of C. trachomatis infection in the United States is notknown, current epidemiological studies indicate that more than 4 millionchlamydial infections occur each year, compared to an estimated 2million gonococcal infections. While all racial, ethnic andsocioeconomic groups are affected, the greatest number of chlamydialinfections occurs among young, 12 to 20 year-old, sexually activeindividuals. Most genitourinary chlamydial infections are clinicallyasymptomatic. Prolonged carriage in both men and women is common. Asmany as 25% of men and 75% of women diagnosed as having chlamydialinfections have no overt signs of infection. As a consequence, theseasymptomatic individuals constitute a large reservoir that can sustaintransmission of the agent within the community.

Far from being benign, serious disease can develop from these infectionsincluding: urethritis, lymphogranuloma venereum (LGV), cervicitis, andepididymitis in males. Ascending infections from the endocervix commonlygives rise to endometritis, pelvic inflammatory disease (PID) andsalpingitis which can cause tubal occlusion and lead ultimately toinfertility in females. Recently, Chlamydia infections have been linkedto heart disease (Bachaier et al. Science:283:1335, 1999; Fan et al.Inf. and Imm. 67:6145, 1999).

C. trachomatis infection of neonates results from perinatal exposure tothe mother's infected cervix. Nearly 70% of neonates born vaginally tomothers with chlamydial cervicitis become infected during delivery. Themucus membranes of the eye, oropharynx, urogenital tract and rectum arethe primary sites of infection. Chlamydial conjunctivitis has become themost common form of ophthalmia neonatorum. Approximately 20-30% ofexposed infants develop inclusion conjunctivitis within 14 days ofdelivery even after receiving prophylaxis with either silver nitrate orantibiotic ointment. C. trachomatis is also the leading cause of infantpneumonia in the United States. Nearly 10-20% of neonates deliveredthrough an infected cervix will develop chlamydial pneumonia and requiresome type of medical intervention.

In developing countries, ocular infections of C. trachomatis causetrachoma, a chronic follicular conjunctivitis where repeated scarformation leads to distortion of the eyelids and eventual loss of sight.Trachoma is the world's leading cause of preventable blindness. TheWorld Health Organization estimates that over 500 million peopleworldwide, including about 150 million children, currently suffer fromactive trachoma and over 6 million people have been blinded by thisdisease.

In industrialized countries, the costs associated with treatingchlamydial infections are enormous. In the United States, the annualcost of treating these diseases was estimated at $2.5-3 billion in 1992and has been projected to exceed $8 billion by the year 2000.

One potential solution to this health crisis would be an effectivechlamydial vaccine. Several lines of evidence suggest that developing aneffective vaccine is feasible.

Studies in both humans and primates have shown that short-termprotective immunity to C. trachomatis can be produced by vaccinatingwith whole Chlamydia. However, protection was characterized as shortlived, serovar specific, and due to mucosal antibody production.Additionally, in some vaccinees disease was exacerbated when theseindividuals became naturally infected with a serovar different from thatused for immunization. This adverse reaction was ultimately demonstratedto be due to a delayed-type hypersensitivity response. Thus, the needexists to develop a subunit-based chlamydial vaccine capable ofproducing an efficacious but nonsensitizing immune response. Such asubunit vaccine may need to elicit both mucosal neutralizing secretoryIgA antibody and/or cellular immune response to be efficacious.

Subunit vaccine development efforts to date have focused almostexclusively on the major outer membrane protein (MOMP). MOMP is anintegral membrane protein of approximately 40 kDa in size and comprisesup to about 60% of the infectious elementary body (EB) membrane protein(Caldwell et al. 1981. Infect. Immun., 31:1161-1176). MOMP impartsstructural integrity to the extracellular EB and is thought to functionas a porin-like molecule when the organism is growing intracellularlyand is metabolically active. With the exception of four surface exposedvariable domains (VDI-VDIV), MOMP is highly conserved among all 18serovars. MOMP is highly immunogenic and can elicit a local neutralizinganti-Chlamydia antibody. However, problems exists with this approach.

To date, most MOMP-specific neutralizing epitopes that have been mappedare located within the VD regions and thus give rise only toserovar-specific antibody. Attempts to combine serovar-specific epitopesin various vaccine vectors (e.g., poliovirus) to generate broadlycross-reactive neutralizing antibodies have been only marginallysuccessful (Murdin et al. 1993. Infect. Immun., 61:4406-4414; Murdin etal. 1995. Infect. Immun., 63:1116-1121).

Two other major outer membrane proteins in C. trachomatis, the 60 kDaand 12 kDa cysteine-rich proteins, as well as the surface-exposedlipopolysaccharide, are highly immunogenic but, unlike MOMP, have notbeen shown to induce a neutralizing antibody (Cerrone et al., 1991,Infect Immun., 59:79-90). Therefore, there remains a need for a novelsubunit-based chlamydial vaccine.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an indication thatsuch reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

This invention is directed to PMP proteins (referred to hereafter and inthe claims as PMP polypeptides or PMP proteins) from Chlamydia spp. Moreparticularly, the present invention encompasses the family of PMPE andPMPI polypeptides of Chlamydia trachomatis and other Chlamydia spp.,including but not limited to, Chlamydia pneumonia, Chlamydia pecorum,and Chlamydia psittaci, having a molecular weight of 90 to 115 kD, andan amino acid sequence of SEQ ID NO:2 or 73 (PMPE) or SEQ ID NO.:4(PMPI) or a sequence homologous thereto, in isolated, purified orrecombinantly produced form. SEQ ID NOs.:2 and 4 represent the aminoacid sequences of the Chlamydia trachomatis L2 serovar PMPE and PMPIproteins, respectively, encoded by the pmpE and pmpI genes. SEQ ID NO:73represents the amino acid sequence of the Chlamydia trachomatis L2serovar PMPE protein encoded by plasmid M15 pREP (pQE-PmpE-Ct#37), whichis derived from a different strain than is SEQ ID NO:2. Preferably, thePMPE and PMPI polypeptides of the invention are encoded by a nucleicacid comprising a nucleotide sequence that hybridizes under low,moderate or, more preferably, highly stringent conditions to a nucleicacid comprising a nucleotide sequence encoding the amino acid sequenceof SEQ ID NO:2, 4, or 73 or a nucleotide sequence of SEQ ID NO.:1,3, or73. Also, preferably, the PMPE and PMPI polypeptides of the invention donot cross react with or bind specifically to the monoclonal antibodysecreted by hybridoma ATCC No. HB10861 available from the American TypeTissue Collection (ATCC).

The nucleotide sequences for the pmpE and pmpI Chlamydia trachomatis L2serovar coding regions are SEQ ID NOs.:1 and 3, respectively. Thenucleotide sequence of the portion of plasmid M15 pREP (pQE-PmpE-Ct#37)encoding the PMPE polypeptide also of the Chlamydia trachomatis L2serovar is SEQ ID NO:72. The present invention encompasses all ChlamydiaPMPE and PMPI polypeptides, particularly those of the Chlamydiatrachomatis L2 serovar, and also including PMPI and PMPE polypeptidesfrom other Chlamydia trachomatis serovars and other Chlamydia species.Identification of these homologs can be accomplished by methods wellknown in the art, for example, but not limited to, nucleic acidhybridization and PCR based techniques. The present inventionencompasses isolated and/or purified PMPE and PMPI polypeptides, andpolypeptides derived therefrom (“PMP-derived polypeptides”, e.g.,derivatives, fragments and analogs thereof), preferably, that elicit animmune reaction against whole Chlamydia cells and/or are specificallybound by antibodies raised against polypeptides having an amino acidsequence of SEQ ID NO.: 2, 4, or 73. The invention further comprisesmethods for making said PMP polypeptides and PMP-derived polypeptides.

Preferably, the PMP protein has the amino acid sequence of SEQ ID NO.:2,4 or 73 or is homologous to any of SEQ ID NO.:2, 4-34, or 73, preferablyhaving an amino acid sequence identity of at least 70% or 80%, morepreferably greater than 90%, and most preferably greater than 95% or99%. These proteins preferably elicit an immune reaction against wholeChlamydia cells and/or are specifically bound by antibodies raisedagainst polypeptides having an amino acid sequence of SEQ ID NO.: 2, 4,or 73. Preferred fragments of the protein comprise an amino acidsequence of any of SEQ ID NOs.:5-34.

Preferably, the PMP protein is an outer membrane protein. Morepreferably, the PMP protein is surface localized. Preferably, the PMPprotein has at least one GGAI (Gly Gly Ala Ile) domain. It is intendedthat PMP proteins from all species of Chlamydia are included in thisinvention; however, preferred species include Chlamydia trachomatis,Chlamydia psittaci, Chlamydia pecorum and Chlamydia pneumoniae.

The invention also provides PMP fusion peptides having B and/or T-cellstimulating activity, preferably comprising at least two T or B cellepitopes derived from the same or from different Chlamydia PMP proteinswhich proteins, or portions thereof, are arranged in a contiguouspolypeptide in a configuration different from a naturally occurringconfiguration of the regions of a Chlamydia PMP protein.

A preferred polypeptide of the invention is a fusion polypeptidecomprising at least two peptides, said at least two peptides, eachconsisting of amino acid sequences selected from the group consisting ofSEQ ID NOs.:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34, with theproviso that the peptides of the polypeptide are arranged in acontiguous polypeptide configuration that is different from theconfiguration of a naturally occurring PMPE or PMPI polypeptide (e.g.,is not a naturally occurring PMPE or PMPI polypeptide or fragmentthereof).

Other preferred PMP-derived polypeptides of the invention are isolatedor purified fusion polypeptides wherein the polypeptide comprises one ormore of the amino acid sequences of SEQ ID NO.:5, 6, 7, 8, 9, 10 or 11or an isolated or purified fusion polypeptide wherein the polypeptidecomprises an amino acid sequence of SEQ ID NO.:23, 24, 25, 26, 27, 28,or 29, with the proviso that the peptides of the polypeptide arearranged in a configuration that is different from the configuration ofa naturally occurring PMPE or PMPI polypeptide. A preferred PMP-derivedpolypeptide is an isolated or purified fusion polypeptide, wherein thepolypeptide comprises an amino acid sequence of SEQ ID NO.:5, 6, 7, 8,9, 10, 11, 23, 24, 25, 26, 27, 28 or 29, with the proviso that thepeptides are arranged in a configuration that is different from theconfiguration of a naturally occurring PMPE or PMPI polypeptide.

Preferably, the PMP-derived polypeptides of the invention areimmunologically cross-reactive with the PMP protein from which they arederived and are capable of eliciting in an animal an immune response toChlamydia. A preferred PMP polypeptide or PMP-derived polypeptide of theinvention induces IgM, IgG, IgA, and/or IgE antibodies, delayedhypersensitivity T-cell responses and/or cytotoxic T-cell responses tocells expressing Chlamydial antigens (including but not limited to cellsinfected with Chlamydia and antigen presenting cells such asmacrophages, dendritic cells, B cells, or synthetic antigen presentingcells which display Chlamydial antigens), native PMP proteins from whichthe polypeptide is derived, Chlamydia cells, or Chlamydia elementalbodies (EB). In a more preferred embodiment, the PMP polypeptide orPMP-derived polypeptide is capable of eliciting an immune responseagainst other Chlamydia serovars and, more preferably, other Chlamydiaspecies along with the Chlamydia serovar or species in which the PMPpolypeptide occurs naturally.

The invention also encompasses antisera and antibodies, including butnot limited to, cytotoxic or bactericidal polyclonal and monoclonalantibodies, which bind to and are specific for the PMP polypeptide,PMP-derived polypeptides and/or fragments thereof.

Preferably the antibodies bind a PMP protein (preferably a PMPE or PMPIpolypeptide) having the amino acid sequence of SEQ ID NOs.:2, 73, or 4or an amino acid sequence homologous thereto. Also included aremonoclonal antibodies that specifically bind a PMP or PMP-derivedpolypeptide, including but not limited to monoclonal antibodies thatspecifically bind a polypeptide comprising an amino acid sequence of anyof SEQ ID NOs.:2, 4-34 or 73. Also included are antigen bindingfragments of polyclonal or monoclonal antibodies, e.g., Fv, Fab, Fab′and F(ab′)₂ fragments. A further aspect of the invention are chimerizedor humanized antibodies in which one or more of the antigen bindingregions of the anti-PMP antibody is introduced into the framework regionof a heterologous (e.g., human) antibody. In a preferred aspect, theantibodies are human antibodies.

Another aspect of the invention is directed to T-cells raised against anantigenic or immunogenic composition of the invention or T-cellsspecific for antigenic or immunogenic polypeptides of the invention orspecific for cells expressing Chlamydial antigens (including but notlimited to cells infected with Chlamydia or antigen presenting cellspresenting PMP polypeptides such as dendritic cells, B cells, orsynthetic antigen presenting cells), Chlamydia cells, or Chlamydiaelemental bodies (EB).

The invention further provides isolated nucleic acid molecules (DNA orRNA) encoding the PMPE polypeptides, PMPI polypeptides, PMPE-derivedpolypeptides, PMPI-derived polypeptides, vectors comprising saidsequences, host cells containing said vectors or having the sequencesoperably linked to a heterologous promoter, recombinant polypeptidesproduced therefrom, and pharmaceutical compositions comprising thenucleic acid molecules, vectors, and cells.

A preferred aspect of the invention is a nucleotide sequence encoding aPMP protein comprising the amino acid sequence of any of SEQ ID NOs.:2,4-34, or 73 or an amino acid sequence substantially homologous thereto.Also included is an isolated nucleic acid molecule comprising a DNAsequence of SEQ ID NOs.:1, 3 or 72 or a complementary sequence thereof;a fragment of the DNA molecule having the nucleotide sequence of SEQ IDNOs.:1, 3 or 72, or a complementary sequence thereof; or a nucleic acidmolecule which hybridizes under low or moderate stringency conditionsor, more preferably, highly stringent (or stringent) conditions to anyone of the sequences described above. The nucleic acid molecule thathybridizes under stringent conditions preferably has a sequence identityof about 70%, 80%, 90%, 95%, or 99% with any of the sequences identifiedabove, more preferably about 90%.

The invention further encompasses pharmaceutical compositions includingprophylactic or therapeutic compositions, which may be immunogeniccompositions, including vaccines, comprising one or more of the PMPpolypeptides of the invention, optionally in combination with, fused to,or conjugated to one or more other component(s), including a lipid,phospholipid, a carbohydrate, including a lipopolysaccharide, anyproteins either novel or known to those skilled in the art, inactivatedwhole or attenuated organisms, including but not limited to viruses,yeasts, fungi and bacteria. Particularly preferred bacteria include, butare not limited to Neisseria, Chlamydia, Moraxella, Pseudomonas,Streptococcus or Haemophilus bacteria.

In a specific embodiment, the invention encompasses pharmaceuticalcompositions, including prophylactic or therapeutic compositions, whichmay be immunogenic compositions including vaccines, comprising one ormore of the PMP polypeptides and/or PMP-derived polypeptides and anattenuated or inactivated Chlamydia cultivar or an attenuated orinactivated Chlamydia cultivar expressing PMP polypeptide in a greateramount when compared to wild-type Chlamydia.

The invention further encompasses pharmaceutical compositions comprisingisolated nucleic acid molecules encoding PMP polypeptides andPMP-derived polypeptides of the present invention which can be used inmethods to detect Chlamydia infection or to prevent, treat or reduce theseverity of a disease or disorder related to infection with Chlamydia.Such compositions include but are not limited to vectors or recombinanthost cells comprising such nucleic acid molecules or having a nucleotidesequence of the invention operably linked to a heterologous promoter.

The invention also includes diagnostic reagents, that may include anyone or more of the above mentioned aspects, such as native PMP proteins,recombinant PMP proteins, PMP-derived polypeptides, nucleic acidmolecules, immunogenic compositions, antigenic compositions, antisera,T-cells, antibodies, vectors comprising the nucleic acids, andtransformed cells comprising the vectors.

A further aspect of the present invention provides methods fordetermining the presence of nucleic acids encoding a PMP protein or aPMP-derived polypeptide in a test sample, and diagnostic kits andreagents therefor, for determining the presence of a nucleic acidencoding a PMP polypeptide or PMP-derived polypeptide.

Also included in this invention are methods of inducing an immuneresponse to Chlamydia spp. and methods of preventing, treating orameliorating disorders or diseases related to Chlamydia in an animal,including mammals and birds and, preferably, in humans, in need of suchtreatment comprising administering an effective amount of thepharmaceutical or vaccine composition of the invention. Preferreddisorders or diseases include a Chlamydia bacterial infection, includingthose infections that cause trachoma, conjunctivitis, urethritis,lymphogranuloma venereum (LGV), cervicitis, epididymitis, orendometritis, pelvic inflammatory disease (PID), salpingitis, tubalocclusion, infertility, cervical cancer, reactive arthritis,inflammatory heart disease, dilated/cardiomyopathy, autoimmunemyocarditis, or atherosclerosis.

A further aspect of the invention provides antagonists or agonists whichinhibit or enhance, respectively, the activity or expression of thepolypeptides or nucleic acid molecules of the invention. In particularembodiments, the agonists or antagonists kill Chlamydia cells or arrestChlamydia cell growth, i.e., can be used to treat or prevent Chlamydiainfection.

A further aspect of the invention is a method for identifying compoundswhich interact with and inhibit or activate an activity of thepolypeptides or nucleic acid molecules of the invention comprisingcontacting a composition comprising the polypeptide or the nucleic acidmolecule with the compound to be screened under conditions that permitinteraction between the compound and the polypeptide or nucleic acidmolecule to assess the interaction of a compound and to detectinteraction of the compound with the polypeptide of nucleic acid. Theinteraction of the compound with the polypeptide or nucleic acidmolecule is determined by the association of a second component (e.g.,an antibody) capable of providing a detectable signal in response to theinteraction of the polypeptide or nucleic acid molecule with thecompound; and determining the presence or absence of a signal generatedfrom the interaction of the compound with the polypeptide or nucleicacid molecule. Alternatively, the interaction of the compound with thepolypeptide or nucleic acid molecule is determined by the ability of thecompound to inhibit the activity of the polypeptide or the nucleic acidmolecule.

3.1. Abbreviations

-   -   anti-PMP=PMP polypeptide antibody or antiserum    -   ATCC=American Type Culture Collection    -   immuno-reactive=capable of provoking a cellular or humoral        immune response    -   kD or kDa=kilodaltons    -   OG=n-octyl-D-glucopyranoside or octyl glucoside    -   OMP=outer membrane protein    -   OMPs=outer membrane proteins    -   PBS=phosphate buffered saline    -   PAGE=polyactylamide gel electrophoresis    -   polypeptide=a peptide of any length, preferably having eight or        more amino acid residues    -   SDS=sodium dodecylsulfate    -   SDS-PAGE=sodium dodecylsulfate polyacrylamide gel        electrophoresis

Nucleotide sequences defined herein are represented by one-lettersymbols for the bases as follows:

-   -   A (adenine)    -   C (cytosine)    -   G (guanine)    -   T (thymine)    -   U (uracil)    -   M (A or C)    -   R (A or G)    -   W (A or T/U)    -   S (C or G)    -   Y (C or T/U)    -   K (G or T/U)    -   V (A or C or G; not T/U)    -   H (A or C or T/U; not G)    -   D (A or G or T/U; not C)    -   B (C or G or T/U; not A)    -   N (A or C or G or T/U) or (unknown)

Peptide and polypeptide sequences defined herein are represented byone-letter symbols for amino acid residues as follows:

-   -   A (alanine)    -   R (arginine)    -   N (asparagine)    -   D (aspartic acid)    -   C (cysteine)    -   Q (glutamine)    -   E (glutamic acid)    -   G (glycine)    -   H (histidine)    -   I (isoleucine)    -   L (leucine)    -   K (lysine)    -   M (methionine)    -   F (phenylalanine)    -   P (proline)    -   S (serine)    -   T (threonine)    -   W (tryptophan)    -   Y (tyrosine)    -   V (valine)    -   X (unknown)

The present invention may be more fully understood by reference to thefollowing detailed description of the invention, non-limiting examplesof specific embodiments of the invention and the appended figures.

4. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Schematic map of the C. trachomatis PMPE expression plasmid M15pREP (pQE-PmpE-Ct#37). The mature form of the C. trachomatis PMPEprotein is expressed in E. coli as a fusion protein carrying theMRGS-(H)₆ domain encoded by the vector plasmid pQE-30 at the N-terminus.

FIG. 2. Schematic map of the C. trachomatis PMPI expression plasmidTOP10 (pBAD-pmpI-Ct-Uni#7). The C. trachomatis PMPI protein is expressedin E. coli as a fusion protein carrying the HP-Thio domain encoded bythe vector plasmid pBAD/Thio-E at the N-terminus.

FIG. 3. A Coomassie blue-stained SDS-PAGE gel of the gel-purified C.trachomatis PMPE protein expressed from the pQE-pmpE-Ct #37 plasmid. ThePMPE protein migrates as an ˜100 kDa protein. Pre-stained molecularweight markers (Lane 1) are Myosin (˜250 kDa), Phosphorylase B (˜148kDa), BSA (˜98 kDa), GDH (˜64 kDa), ADH (˜50 kDa), CAH (˜36 kDa),Myoglobulin (30 kDa), Lysozyme (16 kDa), Aprotinin (˜6 kDa), Insulin Bchain (˜4 kDa) (See Blue prestained standard Novex LC5625).

FIG. 4. A Coomassie blue-stained SDS-PAGE gel of E. coli Top10 cellextracts carrying the C. trachomatis PMPI expression plasmidpBAD-pmpI-ct-Uni#7. Lane 1, pre-stained molecular weight markers (NovexMultiMark LC5725); lane 2, non-induced cells; lane 3, arabinose inducedcells. The C. trachomatis PMPI protein in the arabinose-induced lane isindicated by an arrow. Molecular weight markers (Lane 1) are Myosin(˜250 kDa), Phosphorylase B (˜148 kDa), GDH (˜60 kDa), CAH (˜42 kDa),Myoglobulin-Blue (˜30 kDa), Myoglobulin-Red (˜22 kDa), Lysozyme (˜17kDa).

FIGS. 5A-E. Full length nucleotide sequence and corresponding deducedamino acid sequence of the PMPE polypeptide of Chlamydia trachomatis L2(SEQ ID NOs.:1 and 2).

FIGS. 6A-D. Full length nucleotide sequence and corresponding deducedamino acid sequence of the PMPI polypeptide of Chlamydia trachomatis L2(SEQ ID NOs.:3 and 4).

FIG. 7. In vitro antigen-specific spleen cell proliferative response inanimals immunized with recombinant PMPE and an adjuvant. Spleens wereharvested from immunized C3H HeOuJ female mice approximately 14 daysafter a three dose immunization regimen. Single cell suspensions wereprepared and the cells from two animals were pooled for analysis.Aliquots of the pooled cell samples were incubated for 72-96 hours inthe presence of a test stimulant and pulsed with ³H-thymidine for thelast 18-24 hours. Pooled samples incubated and pulsed labeled inparallel but in the absence of any stimulant served as the baseline³H-thymidine uptake control. Represented is the stimulation index ofcells stimulated with ConA (concanavalin A) (positive stimulationcontrol); adjuvant (adjuvant employed in immunizations); PMPE(recombinant pmpE protein); EB (UV-inactivated C. trachomatis elementarybodies). 1 μg/ml (open bars), 4 μg/ml (solid bars) and 8 μg/ml (hatchedbars) denote the three concentrations of in vitro stimulant used in theexperiment. Stimulation index (SI) denotes the difference in³H-thymidine incorporation of stimulated cells minus the backgroundincorporation of the unstimulated controls. Bars denote themean±standard deviation in SI.

FIGS. 8A-D. Full length nucleotide and corresponding deduced amino acidsequence of the PMPE polypeptide of Chlamydia trachomatis serovar L2contained in plasmid M15pREP (PQE-PmpE-Ct#37) (SEQ ID Nos.:72 and 73).

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. Chlamydia PMP Polypeptides

The present invention is generally directed to compositions and methodsfor the diagnosis, prevention, and treatment of Chlamydial infection. Inone aspect, the composition of the subject invention provides purenative (wildtype) or recombinantly produced PMP polypeptides thatcomprise at least one immunogenic portion of a Chlamydia antigen.

The terms “treatment” or “therapy” as used herein and in the claimsencompasses elimination, reduction or amelioration of disease symptomscaused directly or indirectly by the organism or numbers of organismspresent.

In particular embodiments, the term “Chlamydia” refers to any Chlamydialspecies (spp.) including but not limited to Chlamydia trachomatis,Chlamydia pneumonia, Chlamydia psittaci and Chlamydia pecorum.

Strains from any of these organism may be obtained worldwide from anybiologicals depository, particularly strains of Chlamydia ATCC VR-346,VR-347, VR-348B, VR-571B, VR-572, VR-573, VR-577, VR-578, VR-878,VF-879, VP-880, VR-885, VR-886, VR-887, VR-901B, VR-902B, VR-903,VR-1355, VR-1474, VR-1477, VR-2282, which may be obtained from the ATCC.

In a particular embodiment, the Chlamydia PMP protein is a PMPEpolypeptide comprising or consisting of the amino acid sequence of SEQID NO.:2 or SEQ ID NO:73. In another embodiment, the PMPE polypeptidecomprises residues 32-965 of SEQ ID NO:2 or residues 23-956 of SEQ IDNO:73

In another particular embodiment, the PMPE polypeptide is encoded by thenucleotide sequence of SEQ ID NO.:1 or SEQ ID NO:72. In anotherembodiment, the Chlamydia PMPE polypeptide comprises or consists of anamino acid sequence which is homologous to SEQ ID NO.:2 or 73, or aportion thereof or is encoded by a nucleotide sequence homologous to(for example, that hybridizes under low, moderate or high stringencyconditions to) the nucleotide sequence of SEQ ID NO.:1 or 72, or aportion thereof.

In another particular embodiment, the Chlamydia PMP protein is a PMPIprotein comprising or consisting of the amino acid sequence of SEQ IDNO.:4. In another embodiment, the Chlamydia polypeptide is a PMPIpolypeptide encoded by the nucleotide sequence of SEQ ID NO.:3 or thenucleotide sequence (specifically, the PMPI encoding portion) of plasmidTOP10 (pBAD-pmpI-Ct-Uni)#7. In another embodiment, the Chlamydiapolypeptide comprises or consists of an amino acid sequence which ishomologous to SEQ ID NO.:4, or a portion thereof, or is encoded by anucleotide sequence homologous to (for example, that hybridizes underlow, moderate or high stringency conditions to) the nucleotide sequenceof SEQ ID NO.:3, or a portion thereof.

The present invention provides the family of Chlamydia PMPE and PMPIproteins. The amino acid sequences of SEQ ID NOs.:2, 4, and 73 and thenucleotide sequences of SEQ ID NOs.:1, 3, and 72 represent the aminoacid and nucleotide sequences, respectively, of the PMPE and PMPIpolypeptides, respectively, of the Chlamydia trachomatis L2 serovar (SEQID NOs.:72 and 73 being variants from a different strain of theChlamydia trachomatis L2 serovar than SEQ ID NOs.:1-4). The inventionalso relates to PMPE and PMPI polypeptides from other Chlamydiatrachomatis serovars and strains and other Chlamydia species, as well asother derivatives and fragments thereof (i.e., PMPE- and PMPI-derivedpolypeptides). In a preferred embodiment, immunization with thepolypeptide of the invention elicits antibodies that specifically bindto PMPE or PMPI polypeptides from other Chlamydia serovars and,preferably, species (preferably all species, but may be a subset ofspecies) besides the serovar and species from which the polypeptide wasisolated or derived.

As used herein a “homologous” sequence is at least 70%, preferablygreater than 80%, more preferably greater than 90%, most preferably 95%or 99% identical in sequence to a reference amino acid or nucleotidesequence of identical size or when compared to a reference sequence whenthe alignment or comparison is conducted by a computer homology programor search algorithm known in the art. By way of example and notlimitation, useful computer homology programs include the following:Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990, J. ofMolec. Biol., 215:403-410, “The BLAST Algorithm”; Altschul et al., 1997,Nuc. Acids Res. 25:3389-3402) a heuristic search algorithm tailored tosearching for sequence similarity which ascribes significance using thestatistical methods of Karlin and Altschul 1990, Proc. Nat'l Acad. Sci.USA, 87:2264-68; 1993, Proc. Nat'l Acad. Sci. USA 90:5873-77. Fivespecific BLAST programs perform the following tasks:

1) The BLASTP program compares an amino acid query sequence against aprotein sequence database.

2) The BLASTN program compares a nucleotide query sequence against anucleotide sequence database.

3) The BLASTX program compares the six-frame conceptual translationproducts of a nucleotide query sequence (both strands) against a proteinsequence database.

4) The TBLASTN program compares a protein query sequence against anucleotide sequence database translated in all six reading frames (bothstrands).

5) The TBLASTX program compares the six-frame translations of anucleotide query sequence against the six-frame translations of anucleotide sequence database.

Smith-Waterman (Smith-Waterman, 1981, J. of Molec. Biol., 147:195-197)is a mathematically rigorous algorithm for sequence alignments.

FASTA (see Pearson et al., 1988, Proc. Nat'l Acad. Sci. USA,85:2444-2448) is a heuristic approximation to the Smith-Watermanalgorithm.

By further way of example and not limitation, useful computer homologyalgorithms and parameters for determining percent identity include thefollowing:

To determine the percent identity of two amino acid sequences or of twonucleotide sequences, e.g., between PMP sequences and other knownsequences, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment, the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, a preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Nat'lAcad. Sci. USA, 87:2264-68; as modified by 1993, Proc. Nat'l Acad. Sci.USA 90:5873-77. Such algorithm is incorporated into the NBLAST andXBLAST programs of Altschul, 1990, J. of Molec. Biol., 215:403-410.BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12 to obtain nucleotide sequences homologous to anucleic acid molecule of the invention. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to a protein molecule of the invention.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul, 1997, Nuc. Acids Res. 25:3389-3402.Alternatively, PSI-BLAST can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters ofthe respective programs can be used. Another preferred, non-limitingexample of a mathematical algorithm utilized for the comparison ofsequences is the algorithm of Myers and Miller, CABIOS (1989). Such analgorithm is incorporated into the ALIGN program (version 2.0) which ispart of the CGC sequence alignment software package. When using theALIGN program for comparing amino acid sequences, a PAM120 weightresidue table a gap length penalty of 12, and a gap penalty of 4 can beused. Additional algorithms for sequence analysis are known in the artand include ADVANCE and ADAM as described in Torellis and Robotti (1994)Comput. Appl. Biosc., 10:3-5; and FASTA described in Pearson and Lipman,1988, Proc. Nat'l Acad. Sci. USA, 85:2444-2448. Within FASTA, ktup is acontrol option that sets the sensitivity and speed of the search. Ifktup=2, similar regions in the two sequences being compared are found bylooking at pairs of aligned residues; if ktup=1, single aligned aminoacids are examined. Ktup can be set to 2 or 1 for protein sequences, orfrom 1 to 6 for nucleotide sequences. The default, if ktup is notspecified, is 2 for proteins and 6 for nucleotides. Alternatively,protein sequence alignment may be carried out using the CLUSTAL Walgorithm as described by Higgins et al., 1996, Methods Enzymol.,266:383-402.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

According to various aspects of the invention, the polypeptides of theinvention are characterized by their apparent molecular weights based onthe polypeptides' migration in SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) relative to the migration ofknown molecular weight markers. While any molecular weight standardsknown in the art may be used with the SDS-PAGE, preferred molecularweight markers comprise pre-stained Myosin (250 kDa), Phosphorylase B(148 kDa), BSA (98 kDa) and GDH (64 kDa). One skilled in the art willappreciate that the polypeptides of the invention may migratedifferently in different types of gel systems (e.g., different buffers;different types and concentrations of gel, crosslinkers or SDS, etc.).One skilled in the art will also appreciate that the polypeptides mayhave different apparent molecular weights due to different molecularweight markers used with the SDS-PAGE. Hence, the molecular weightcharacterization of the polypeptides of the invention is intended to bedirected to cover the same polypeptides on any SDS-PAGE system and withany set of molecular weight markers which might indicate slightlydifferent apparent molecular weights for the polypeptides than thosedisclosed herein.

In specific embodiments, the subject invention discloses PMPpolypeptides comprising an immunogenic portion of a Chlamydia antigen,wherein the Chlamydia antigen comprises an amino acid sequence encodedby a nucleic acid molecule comprising a sequence selected from the groupconsisting of (a) nucleotide sequences of SEQ ID NO.:1, 3 and 72; (b)the complements of said nucleotide sequences; and (c) variants of suchsequences.

5.2. Chlamydia PMP-Derived Polypeptides

The term “antigens” and its related term “antigenic” as used herein andin the claims refers to a substance to which an antibody or T-cellreceptor specifically binds. As used herein, antisera, antibodies andT-cells are “antigen-specific” if they specifically bind to or reactwith an antigen and do not react detectably with unrelated proteinsother than by non-specific interaction. Preferably said antigens areimmunogenic.

The term “immunogenic” as used herein and in the claims refers to theability to induce an immune response, e.g., an antibody and/or acellular immune response in an animal, preferably a mammal or a bird.

In a specific embodiment of the invention, proteins consisting of orcomprising a fragment of a PMPE protein consisting of at least 8(contiguous) amino acids of the protein are provided. In otherembodiments, the fragment consists of at least 5, 10, 20, 40, 50, 60,80, 100, 150, 200, 300, 400 or 500 amino acids of SEQ ID NO.:2 or 73 ora sequence homologous thereto. In specific embodiments, such fragmentsare not larger than 10, 11, 12, 15, 20, 25, 35, 50, 75, 80, 90, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675 or 700 amino acids. In preferredembodiments, the PMPE-derived polypeptide contains a sequence forming anouter-surface epitope, i.e., a portion of the peptide located on theoutside of the Chlamydia cell and that can be bound by anti-PMPEantibodies.

In a particular embodiment, the PMPE-derived polypeptide is a fragmentof a PMPE polypeptide which fragment comprises or consists of an aminoacid sequence of any of SEQ ID NOs.:5-22. In another particularembodiment, the PMPE-derived polypeptide is a fragment of a PMPEpolypeptide, which fragment comprises or consists of an amino acidsequence of any of SEQ ID NOs.:5-22, but also comprises additionalC-terminal or N-terminal PMPE sequences.

In a specific embodiment of the invention, proteins are provided thatconsist of or comprise a fragment of a PMPI protein consisting of atleast 8 (continuous) amino acids of SEQ ID NO.:4 or a sequencehomologous thereto. In other embodiments, the fragment consists of atleast 5, 10, 15, 20, 25, 50, 75, 100, 150 or 200 amino acids of SEQ IDNO.:4. In specific embodiments, such fragments are not longer than 10,11, 12, 15, 20, 25, 35, 50, 75, 80, 90, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 400, 425, 450, 475, 500, 525, 550, 575, 600,625, 650, 675 or 700 amino acids. In preferred embodiments, PMPI-derivedpolypeptide contains a sequence forming an outer-surface epitope.

In a particular embodiment, the PMPI-derived polypeptide is a fragmentof a PMPI peptide which comprises the amino acid sequence of any of SEQID NOs.:23-34.

In another particular embodiment, the PMPE-derived polypeptide is afragment of a PMPE polypeptide which comprises the amino acid sequenceof any of SEQ ID NOs.:5-22, but also comprises additional C-terminal orN-terminal PMPI sequences.

Preferably, the PMPE-derived polypeptides of the invention areimmunologically cross-reactive with the PMPE polypeptide, and arecapable of eliciting in an animal an immune response to Chlamydia,Chlamydia elemental bodies (EB), Chlamydia reticulate bodies (RBs),Chlamydia infected cells or antigen presenting cells expressingChlamydial antigens and/or are able to be bound by anti-PMPE antibodies.

Preferably, the PMPI-derived polypeptides of the invention areimmunologically cross-reactive with the PMPI polypeptide, and arecapable of eliciting in an animal an immune response to Chlamydia,Chlamydia elemental bodies (EB), reticulocyte bodies (RBs), Chlamydiainfected cells or antigen presenting cells expressing Chlamydialantigens and/or are able to be bound by anti-PMPI antibodies. Morepreferably, the PMP-derived polypeptides of the invention comprisesequences forming one or more epitopes of the native PMPE or PMPIpolypeptide of Chlamydia (i.e., the epitopes of PMPE or PMPI polypeptideas they exist in intact Chlamydia cells). Such preferred PMPE-derived orPMPI-derived polypeptides can be identified by their ability tospecifically bind polyclonal or monoclonal antibodies raised to intactChlamydia cells (e.g., antibodies elicited by formaldehyde orglutaraldehyde fixed Chlamydia cells; such antibodies are referred toherein as “anti-whole cell” antibodies). For example, peptides from alimited or complete protease digestion of the PMPE or PMPI polypeptideare fractionated using standard methods and tested for their ability tobind anti-whole cell antibodies. Reactive polypeptides are isolated andtheir amino acid sequence determined by methods known in the art. In apreferred embodiment, the PMPE and/or PMPI-derived polypeptide comprisesone or more portions of a PMPE or PMPI protein, or derivative thereof,that is a T-cell epitope.

Preferably, the PMP polypeptides and PMP-derived polypeptides of theinvention are not bound specifically by the monoclonal antibody secretedby hybridoma ATCC No. HB10861 available from the ATCC.

PMP-derived polypeptides can also be constructed by making deletionsthat remove a part of the parent polypeptide, while retaining thedesired specific antigenicity and/or immunogenicity. Deletions can alsoremove regions of high variability among strains.

Also preferably, the PMP-derived polypeptides of the invention comprisesequences that form one or more epitopes of a native PMP polypeptide,which epitopes elicit bactericidal or opsonizing antibodies. Suchpreferred PMP-derived polypeptides may be identified by their ability togenerate antibodies that kill Chlamydia spp., particularly, Chlamydiatrachomatis cells. For example, polypeptides from a limited or completeprotease digestion or chemical cleavage of a PMP polypeptide arefractionated using standard methods (e.g., by limited proteolyticdigestion using enzymes such as trypsin, papain, or related proteolyticenzymes or by chemical cleavage using agents such as cyanogen bromideand followed by fractionation of the digestion or cleavage products),injected into animals, and the antibodies produced therefrom are testedfor the ability to interfere with or kill Chlamydia cells and/orChlamydia infected cells. Once identified and isolated, the amino acidsequences of such preferred PMP-derived polypeptides are determinedusing standard sequencing methods. The determined sequence may be usedto enable production of such polypeptides by synthetic chemical and/orgenetic engineering means.

These preferred PMP-derived polypeptides also can be identified by usinganti-whole cell antibodies to screen bacterial libraries expressingrandom fragments of Chlamydia genomic DNA or cloned nucleotide sequencesencoding a PMPE or PMPI polypeptide or fragments thereof. See, e.g.,Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., Vol. 1, Chapter 12.The reactive clones are identified and their inserts are isolated andsequenced to determine the amino acid sequences of such preferredPMP-derived polypeptides.

Examples of immunogenic portions of antigens contemplated by the presentinvention include polypeptides comprising or consisting of the fragmentsset forth in Tables 1 and 2, where the numbers following the PMPE (Table1, column 1) or PMPI (Table 2, column 1) designation refer to the aminoacid residues in SEQ ID NOs.:2 or 4, respectively. Polypeptidescomprising at least an immunogenic portion of one or more Chlamydiaantigens or immunogenic portions as described herein may generally beused, alone or in combination, to detect, prevent, treat or reduce theseverity of Chlamydial infection. TABLE 1 PREFERRED FRAGMENTS OF PMPEFRAGMENT SEO ID NO.: PMPE15-56  5 PMPE15-121  6 PMPE45-125  7PMPE125-190  8 PMPE195-261  9 PMPE275-366 10 PMPE375-440 11 PMPE440-49012 PMPE525-590 13 PMPE590-625 14 PMPE615-650 15 PMPE625-700 16PMPE725-800 17 PMPE755-775 18 PMPE785-845 19 PMPE815-865 20 PMPE1-31 21PMPE1-500 22

TABLE 2 PREFERRED FRAGMENTS OF PMPI FRAGMENT SEQ ID NO.: PMPI 13-40 23PMPI 70-110 24 PMPI 150-225 25 PMPI 250-290 26 PMPI 370-455 27 PMPI400-455 28 PMPI 470-520 29 PMPI 615-670 30 PMPI 710-775 31 PMPI 765-82532 PMPI 830-860 33 PMP 1-500 34

Polypeptides having a sequence homologous to one of the PMP polypeptidesof the invention, including naturally-occurring allelic variants, aswell as mutants, variants or any other non-naturally occurring variants,preferably those that cross-react with antibodies against a PMPpolypeptide of the present invention, are encompassed by the presentinvention.

Allelic variants are very common in nature. For example, a bacterialspecies e.g., C. trachomatis, is usually represented by a variety ofstrains or serovars that differ from each other by minor allelicvariations. Indeed, a polypeptide that fulfills the same biologicalfunction in different strains can have an amino acid sequence that isnot identical in each of the strains. Such an allelic variation may beequally reflected at the nucleic acid molecule level.

An allelic variant is an alternate form of a polypeptide that ischaracterized as having a substitution, deletion, or addition of one ormore amino acids that does not substantially alter the biologicalfunction of the polypeptide. By “biological function” is meant thefunction of the polypeptide in the cells in which it naturally occurs,even if the function is not necessary for the growth or survival of thecells.

Nucleic acid molecules, e.g., DNA molecules, encoding allelic variantscan easily be retrieved by the polymerase chain reaction (PCR)amplification of genomic bacterial DNA extracted by conventionalmethods. This involves the use of synthetic oligonucleotide primersmatching upstream and downstream sequences of the 5′ and 3′ ends of theencoding domains. Typically, a primer can consist of 10 to 40,preferably 15 to 25 nucleotides. It may be also advantageous to selectprimers containing C and G nucleotides in a proportion sufficient toensure efficient hybridization; e.g., an amount of C and G nucleotidesof at least 40%, preferably 50% of the total number of nucleotides inthe primer.

Variants of Chlamydia trachomatis PMP proteins, such as PMP proteinsfrom the A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, MoPN, L1, L2, andL3 serovars, which share sequence homology to the PMP polypeptides andnucleic acid molecules described herein are also provided.

“Homolog” or “homologous” is defined as being at least 70, 80, 85, 90,95 or 99% identical to a reference sequence of identical size or whenthe alignment or comparison is by a computer homology program or searchalgorithm known in the art (see Section 5.1 supra). Preferably, theserovar homologs show 70, 80, 85, 90, 95 or 99% identity to thecorresponding polypeptide sequence or sequences described herein. Mostpreferably, the serovar homologs show 95-99% homology to thecorresponding polypeptide sequence or sequences described herein. Also,homologous nucleotide sequences exhibit 70, 80, 85, 90, 95 or 99%identity to the corresponding nucleotide sequence or sequences describedherein.

A PMP-derived polypeptide of the invention may also be a modified PMPEor PMPI polypeptide or fragment thereof (i.e., a Chlamydia PMPpolypeptide or fragment having one or more amino acid substitutions,insertions and/or deletions of the wild-type Chlamydia PMP sequence oramino acids chemically modified in vivo or in vitro). Such modificationsmay enhance the immunogenicity of the resultant PMP-derived polypeptideproduct or have no effect on such activity.

As used herein, the term “enhance the immunogenicity” refers to anincreased antibody titer or increased cellular immune response elicitedby exposure to the modified polypeptide as compared to the immuneresponse elicited by unmodified polypeptides or formalin orglutaraldehyde fixed Chlamydia. Modification techniques that may be usedinclude, but are not limited to, those disclosed in U.S. Pat. No.4,526,716.

As an illustrative, non-limiting example, one or more amino acidresidues within the PMP-derived polypeptide sequence can be substitutedby another amino acid of a similar polarity which acts as a functionalequivalent, resulting, for example, in a silent alteration. Substitutesfor an amino acid within the sequence may be selected from other membersof the class to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

Included within the scope of the invention are PMP-derived polypeptideswhich are Chlamydia PMP polypeptide fragments or other derivatives oranalogs which are differentially modified during or after translation,e.g., by glycosylation, acetylation, phosphorylation, lipidation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications may be carried outby known techniques, including but not limited to, specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH_(4,) acetylation, formylation, oxidation, reduction,metabolic synthesis in the presence of tunicamycin, etc.

Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the PMPpolypeptide sequence. Non-classical amino acids include but are notlimited to the D-isomers of the common amino acids, α-amino isobutyricacid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl aminoacids, PNA's and amino acid analogs in general. Furthermore, the aminoacid can be D (dextrorotary) or L (levorotary).

A Chlamydia PMP-derived polypeptide may further be a chimericpolypeptide comprising one or more heterologous polypeptides, lipids,phospholipids or lipopolysaccharides of Chlamydia origin or of anotherbacterial or viral origin, fused (e.g., covalently bound) to theamino-terminus or carboxyl-terminus of or is within a complete PMPE orPMPI polypeptide or PMP-derived polypeptide. Useful heterologouspolypeptides to be included within such a chimeric polypeptide include,but are not limited to, a) pre- and/or pro-sequences that facilitate thetransport, translocation and/or processing of the PMP-derivedpolypeptide in a host cell, b) affinity purification sequences, and c)any useful immunogenic sequences (e.g., sequences encoding one or moreepitopes of a surface-exposed protein of a microbial pathogen). Onepreferred heterologous protein of the chimeric polypeptide includesHin47 (see U.S. Pat. Nos. 5,679,547 and 5,721,115, which are herebyincorporated by reference in their entirety). Another preferred chimericpolypeptide includes a High Molecular Weight (HMW) protein of Chlamydiaor fragments thereof (see PCT publication WO 99/17741, entitled“Chlamydia Protein, Gene Sequence and Uses Thereof”, which isincorporated by reference herein in its entirety) or Chlamydia MOMP orfragments thereof (see U.S. Pat. No. 5,869,608, which is incorporatedherein by reference in its entirety). The fragments of these proteinspreferably contain an epitope specifically bound by an antibody raisedagainst the protein. A particularly preferred chimeric protein comprisesone or more of: SEQ ID NOs.:5-34, an N-terminal fragment of HMW proteinand a fragment of MOMP. Other preferred chimeric proteins comprisefragments of PMPE, HMW protein, MOMP, PMPH, and C. trachomatis HtrA. Thesequences of C. trachomatis HtrA and C. trachomatis PMPH are disclosedin Stephens et al., 1998, Science 282:754-759 and in Genbank accessionnos. AAC68420 (HtrA) and AE001360 (PMPH), which are all herebyincorporated by reference in their entireties.

PMP-derived polypeptides also include but are not limited to fusionpolypeptides comprising at least two regions derived from one or moreChlamydia proteins, each having T-cell or antibody stimulating activity.The regions may be derived from the same Chlamydia protein or maycomprise one or more regions from more than one Chlamydia protein. Thepolypeptides are arranged in a nonsequential order or noncontiguousorder (e.g., in an order different from the order of the amino acids ofthe native protein). A preferred polypeptide of the invention is afusion polypeptide comprising at least two peptides, each of whichpeptides consists of an amino acid sequence selected from the groupconsisting of SEQ ID NOs.:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34,with the proviso that the peptides are arranged in a configuration thatis different from the configuration of a naturally occurring PMPE orPMPI polypeptide.

Other preferred PMP-derived polypeptides of the invention are fusionpolypeptides wherein the polypeptide comprises all the peptidesconsisting of the amino acid sequences of SEQ ID NOs.:5, 6, 7, 8, 9, 10and 11, or a fusion polypeptide wherein the polypeptide comprises allthe peptides consisting of the amino acid sequences of SEQ ID NOs.:23,24, 25, 26, 27, 28, and 29, with the proviso that the peptides arearranged in a configuration that is different from the configuration ofa naturally occurring PMPE or PMPI polypeptide. A preferred PMP-derivedpolypeptide is a fusion polypeptide comprising a peptide consisting ofan amino acid sequence of any of SEQ ID NOs.:5, 6, 7, 8, 9, 10, 11, 23,24, 25, 26, 27, 28 or 29, with the proviso that the peptides arearranged in a configuration that is different from the configuration ofa naturally occurring PMPE or PMPI polypeptide. In a preferredembodiment, the fusion proteins of the invention are isolated.

Other preferred PMP-derived polypeptides of the invention are fusionproteins comprising one or more peptides comprising or consisting of theamino acid sequences of SEQ ID NOs:5-34, and one or more fragments(preferably, epitope containing fragments) of one or more other C.trachomatis proteins, including but not limited to HMW protein, PMPH,HtrA, and MOMP. Preferred chimeric fusion proteins comprise an aminoterminal fragment of HMW protein and one or more fragments of MOMP.Particularly preferred chimeric PMPE or PMPI polypeptides comprise oneor more fragments of MOMP which comprise or consist of amino acidresidues 273-333, 64-85, 139-160, 224-237, 288-317, 1-200, 64-350,160-350, 240-333 or 80-105 (for sequence and numbering, see Batteiger etal., 1996, Infect. Immun. 64:542-547 and Yuan et al., 1989, Infect.Immun., 57:1040-1049, both of which are hereby incorporated by referencein their entireties). Preferred PMPE or PMPI chimeric polypeptides mayalso comprise fragments of HMW protein which comprises or consists ofresidues 1-500 or residues 23-500 or residues 1-100, 1-200, 1-300, or1-400. The fragments of HMW protein, MOMP, HtrA and PMPH are at least 8,10, 15, 20, 25, 30, 50, 75 or 100 amino acid fragments.

If desired, the amino acid sequences of the regions can be produced andjoined by a linker.

Suitable peptide linker sequences may be chosen based on the followingfactors: (1) their ability to adopt a flexible extended conformation;(2) their ability to adopt a secondary structure that could interactwith functional epitopes of the first and second polypeptides; (3) thelack of hydrophobic or charged residues that might react with thepolypeptide functional epitopes; (4) the ability to increase solubility;and (5) the ability to increase sensitivity to processing byantigen-presenting cells. Such linkers can be any amino acid sequence orother appropriate link or joining agent.

Linkers useful in the invention include linkers comprising a chargedamino acid pair such as KK or RR, linkers sensitive to cathepsin and/orother trypsin-like enzymes, thrombin or Factor X_(a,) or linkers whichresult in an increase in solubility of the polypeptide.

Preferred peptide linker sequences contain Gly, Asn and Ser residues.Amino acid sequences which may be usefully employed as linkers includethose disclosed in Maratea et al. Gene 40:39-46 (1985); Murphy et al.,Proc. Nat. Acad Sci USA 83:8258-8562 (1986); U.S. Pat. No. 4,935,233 andU.S. Pat. No. 4,751,180. The linker sequence may be from 1 to about 50amino acids in length.

Another particular example of fusion polypeptides of the inventionincludes a PMP polypeptide or PMP-derived polypeptide of the inventionfused to a polypeptide having adjuvant activity, such as the subunit Bof either cholera toxin or E. coli heat labile toxin. Another particularexample of a fusion polypeptide encompassed by the invention includes aPMP polypeptide or PMP-derived polypeptide of the invention fused to acytokine (such as, but not limited to, IL-2, IL-4, IL-10, IL-12, orinterferon). A polypeptide of the invention can be fused to the N— orC-terminal end of a polypeptide having adjuvant activity. Alternatively,a polypeptide of the invention can be fused within the amino acidsequence of the polypeptide having adjuvant activity.

Also preferably, the PMP-derived fusion polypeptides of the inventioncomprise sequences that form one or more epitopes of a native ChlamydiaPMP polypeptide that elicit bactericidal or opsonizing antibodies and/orT-cells. Such preferred PMP-derived polypeptides may be identified bytheir ability to generate antibodies and/or T-cells that kill cellsinfected with Chlamydia spp. cells particularly, Chlamydia trachomatiscells.

5.3. Isolation and Purification of PMP Polypeptides and PMP-DerivedPolypeptides

The invention provides isolated PMPE and PMPI polypeptides, PMPE-derivedand PMPI-derived polypeptides. As used herein, the term “isolated” meansthat the product has been removed from other biological materials withwhich it is naturally associated, or free from other biologicalmaterials derived, for example, from a recombinant host cell that hasbeen genetically engineered to express the polypeptide of the invention.As used herein, the term “purified” means that the product issubstantially free of other biological material with which it isnaturally associated, or free from other biological materials derived,for example, from a recombinant host cell that has been geneticallyengineered to express the polypeptide of the invention. That is, apurified PMP polypeptide composition is at least 70-95% pure PMPpolypeptide by weight, preferably at least 75% pure PMP polypeptide byweight, and more preferably at least 95% pure PMP polypeptide by weight,or most preferably 98% or 99% pure PMP polypeptide by weight. Thus, aChlamydia lysate or membrane preparation on an acrylamide gel (with orwithout SDS), including a portion of the gel containing one or moreprotein bands, of a Chlamydia lysate or membrane preparation ofChlamydia is not a purified preparation or composition of PMPE or PMPI,since the gel comprises other Chlamydia proteins and by weight PMPE orPMPI does not constitute at least 70% pure PMP polypeptide by weight,preferably at least 75% pure PMP polypeptide by weight, and morepreferably at least 95% pure PMP polypeptide by weight, or mostpreferably 98% or 99% pure PMP polypeptide by weight of the preparationor composition. However, a preparation of PMPE or PMPI obtained byeluting the PMPE or PMPI band from the acrylamide gel is a purifiedpreparation of PMPE or PMPI.

The PMP polypeptide of the invention may be isolated from a proteinextract, including a whole cell extract of any Chlamydia spp.,including, but not limited to, Chlamydia trachomatis, Chlamydiapneumoniae, Chlamydia pecorum, and Chlamydia psittaci. Strains from anyof these organisms may be obtained worldwide from any biologicalsdepository, for example, strains of ATCC VR-346, VR-347, VR-348B,VR-571B, VR-572, VR-573, VR-577, VR-578, VR-878, VF-879, VR-880, VR-885,VR-886, VR-887, VR-901B, VR-902B, VR-903, VR-1355, VR-1474, VR-1477, orVR-2282 maybe obtained from the American Type Culture Collection.

Another source of the PMP polypeptide is a protein preparation from agene expression system (such as E. coli) engineered to express a clonedsequence encoding a PMP polypeptide or PMP-derived polypeptide (seeSection 5.5, infra).

The PMP polypeptide can be isolated and purified from the sourcematerial using any biochemical technique and approach well known tothose skilled in the art. In one approach, Chlamydia cellular envelopeis obtained by standard techniques and inner membrane, periplasmic andouter membrane proteins are solubilized using a solubilizing compoundsuch as a detergent or hypotonic solution. A preferred detergentsolution is one containing octyl glucopyranoside (OG), sarkosyl orTRITON X100™ (t-octyl phenoxy-polyethoxy-ethanol). A preferredsolubilizing hypotonic solution is one containing LiCl. The PMPpolypeptide is in the solubilized fraction. Cellular debris andinsoluble material in the extract are separated and removed preferablyby centrifugation. The polypeptides in the extract are concentrated,incubated in SDS-containing Laemmli gel sample buffer at 100° C. for 5minutes and then fractionated by electrophoresis in a denaturing sodiumdodecylsulfate (SDS) polyacrylamide gel from about 6% to about 12%, withor without a reducing agent. See Laemmli, 1970, Nature 227:680-685. Theband or fraction identified as a PMP polypeptide, having an apparentmolecular weight of about 90-115 kDa, as described above, may then bepurified directly from the fraction or gel slice containing the PMPpolypeptide. In a preferred embodiment, the PMP polypeptide has anapparent molecular weight of about 90-115 kDa which can be determined bycomparing its migration distance or rate in denaturing SDS-PAGE relativeto the migration of known molecular weight markers such as of myosin(250 kDa), Phosphorylase B (148 kDa), BSA (98 kDa) and GDH (64 kDa)(weights for pre-stained markers).

Another method of purifying PMP polypeptide is by affinitychromatography using anti-PMP antibodies (see Section 5.4). The affinitychromatography may be carried out using either polyclonal or monoclonalanti-PMP antibodies, preferably, monoclonal antibodies. The antibodiesare covalently linked to agarose gels activated by cyanogen bromide orsuccinamide esters (Affi-Gel, BioRad, Inc.) or by other methods known tothose skilled in the art. The protein extract is loaded on the top ofthe gel and is left in contact with the gel for a period of time andunder standard reaction conditions sufficient for PMP polypeptide tobind to the antibody. Preferably, the solid support is a material usedin a chromatographic column. The affinity gel is washed to remove otherproteins and cell materials not bound by the anti-PMP antibody. The PMPpolypeptide is then removed from the antibody to recover the PMPpolypeptide in isolated, or preferably, in purified form.

A PMP-derived polypeptide of the invention can be produced by chemicaland/or enzymatic cleavage or degradation of an isolated or purified PMPpolypeptide. A PMP-derived polypeptide can also be chemicallysynthesized based on the known amino acid sequence of the PMPpolypeptide and, in the case of a chimeric polypeptide, the amino acidsequence of the heterologous polypeptide, by methods well known in theart. See, for example, Creighton, 1983, Proteins: Structures andMolecular Principles, W.H. Freeman and Co., NY.

A PMP-derived polypeptide can also be produced in a gene expressionsystem expressing a recombinant nucleic acid construct comprising asequence encoding a PMP-derived polypeptide. The nucleotide sequencesencoding polypeptides of the invention may be synthesized, and/orcloned, and expressed according to techniques well known to thoseskilled in the art. See, for example, Sambrook, et al., 1989, MolecularCloning A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NY,Chapter 9.

PMP-derived polypeptides of the invention can be fractionated andpurified by the application of standard protein purification techniques,modified and applied in accordance with the discoveries and teachingsdescribed herein. In particular, preferred PMP polypeptides of theinvention, those that form an outer-surface or exposed epitope of thenative PMP polypeptide, may be isolated and purified according to theaffinity procedures disclosed above for the isolation and purificationof a PMP polypeptide (e.g., affinity purification using anti-PMPantibodies).

If desirable, the polypeptides of the invention may be further purifiedusing standard protein or peptide purification techniques including butnot limited to, electrophoresis, centrifugation, gel filtration,precipitation, dialysis, chromatography (including ion exchangechromatography, affinity chromatography, immunoadsorbent affinitychromatography, dye-binding chromatography, size exclusionchromatography, hydroxyapatite chromatography, reverse-phase highperformance liquid chromatography, and gel permeation high performanceliquid chromatography), isoelectric focusing, and variations andcombinations thereof.

One or more of these techniques may be employed sequentially in aprocedure designed to isolate and/or purify the PMP polypeptide or thePMP-derived polypeptides of the invention according to its/theirphysical or chemical characteristics. These characteristics include thehydrophobicity, charge, binding capability, and molecular weight of theprotein. The various fractions of materials obtained after eachtechnique are tested for binding to the PMP receptor or ligand or toanti-PMP antibodies or for functional activity (“test” activities).Those fractions showing such test activity are then pooled and subjectedto the next technique in the sequential procedure, and the new fractionsare tested again. The process is repeated until fractions are obtainedthat have one or more of the above described “test” activities and thatcontain only a single band (or a very predominant band) or entity whensubjected to polyacrylamide gel electrophoresis or chromatography.

5.4. PMP Immunogens and Anti-PMP Antibodies

The present invention provides antibodies that specifically bind a PMPpolypeptide and/or PMP-derived polypeptide. For the production of suchantibodies, isolated or, preferably, purified preparations of a PMPpolypeptide or PMP-derived polypeptide are used as immunogens in animmunogenic composition. The same immunogen can be used to immunize micefor the production of hybridoma lines that produce monoclonal anti-PMPantibodies. In particular embodiments, the immunogen is an isolated orpurified PMP polypeptide or PMP-derived polypeptide from any Chlamydiastrain, including, but not limited to, Chlamydia trachomatis, Chlamydiapneumoniae, Chlamydia pecorum, and Chlamydia psittaci. Particularlypreferred are the strains of Chlamydia trachomatis from the AmericanType Culture Collection (ATCC): VR-346, VR-347, VR-348B, VR-571B,VR-572, VR-573, VR-577, VR-578, VR-878, VF-879, VR-880, VR-885, VR-886,VR-887, VR-901B, VR-902B, VR-903, VR-1355, VR-1474, VR-1477, VR-2282.

In other embodiments, peptide fragments of a PMP polypeptide are used asimmunogens. Preferably, peptide fragments of a purified PMP polypeptideare used. The peptides may be produced by protease digestion, chemicalcleavage of isolated or purified PMP polypeptide, chemical synthesis orby recombinant expression, after which they are then isolated orpurified. Such isolated or purified peptides can be used directly asimmunogens. In particular embodiments, useful peptide fragments are 8 ormore amino acids in length.

Useful immunogens may also comprise such peptides or peptide fragmentsconjugated to a carrier molecule, preferably a carrier protein. Carrierproteins may be any commonly used in immunology, include, but are notlimited to, bovine serum albumin (BSA), chicken albumin, keyhole limpethemocyanin (KLH), tetanus toxoid and the like. For a discussion ofhapten protein conjugates, see, for example, Harlow and Lane Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988, or a standard immunology textbook such as Roitt, etal., IMMUNOLOGY, C.V. Mosby Co., St. Louis, Mo. (1985) or Klein,IMMUNOLOGY, Blackwell Scientific Publications, Inc., Cambridge, Mass.(1990).

In yet another embodiment, for the production of antibodies thatspecifically bind one or more epitopes (preferably, outer membraneepitopes) of a native PMP polypeptide, intact Chlamydia cells orelemental bodies (EBs) or reticulate bodies (RBs) prepared therefrom, orcells infected with Chlamydia are used as immunogen. The cells, EBs, RBsor cells infected with Chlamydia may be fixed with agents such asformaldehyde or glutaraldehyde before immunization. See Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988, Chapter 15. It is preferred that suchanti-whole cell antibodies be monoclonal antibodies. Hybridoma linesproducing the desired monoclonal antibodies can be identified by usingpurified PMP polypeptide, intact Chlamydia cells, EBs, RBs or cellsinfected with Chlamydia as the screening ligand. The immunogen forinducing these antibodies may be whole Chlamydia cells, EBs, RBs,extracts or lysates of any Chlamydia, including, but not limited to,Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia pecorum, andChlamydia psittaci. Particularly preferred are strains of Chlamydiastrains ATCC VR-346, VR-347. VR-348B, VR-571B, VR-572, VR-573, VR-577,VR-578, VR-878, VF-879, VR-880, VR-885, VR-886, VR-887, VR-901B,VR-902B, VR-903, VR-1355, VR-1474, VR-1477, VR-2282.

Polyclonal antisera produced by immunization with cells infected withChlamydia, whole cells, EBs or RBs contain antibodies that bind otherChlamydia proteins (“non-anti-PMP antibodies”) and thus are morecumbersome to use when it is known or suspected that the sample containsother Chlamydia proteins or materials that are cross-reactive with theseother proteins. Under such circumstances, any binding by the anti-wholecell antibodies of a given sample or band must be verified bycoincidental binding of the same sample or band by antibodies thatspecifically bind the PMP polypeptide (e.g., anti-PMPE antibodies oranti-PMPI antibodies) and/or a PMP-derived polypeptide, or bycompetition tests using anti-PMP antibodies, PMP polypeptides orPMP-derived polypeptides as the competitor (i.e., addition of anti-PMPantibodies, PMP polypeptide or PMP-derived polypeptide to the reactionmix lowers or abolishes sample binding by anti-whole cell antibodies).Alternatively, such polyclonal antisera containing “non-anti-PMP”antibodies, may be cleared of such non-anti-PMP antibodies by standardapproaches and methods. For example, the non-anti-PMP antibodies may beremoved by precipitation with cells having a deletion of the PMP codingsequence or Chlamydia strains known not to have the PMP polypeptide; orby absorption to columns comprising such cells or outer membraneproteins of such cells.

In further embodiments, useful immunogens for eliciting antibodies ofthe invention comprise mixtures of two or more of any of theabove-mentioned individual immunogens.

Immunization of animals with the immunogens described herein, preferablyof humans, rabbits, rats, ferrets, mice, sheep, goats, cows or horses,is performed following procedures well known to those skilled in theart, for purposes of obtaining antisera containing polyclonal antibodiesor hybridoma lines secreting monoclonal antibodies.

Monoclonal antibodies can be prepared by standard techniques, given theteachings contained herein. Such techniques are disclosed, for example,in U.S. Pat. No. 4,271,145 and U.S. Pat. No. 4,196,265. Briefly, ananimal is immunized with the immunogen. Hybridomas are prepared byfusing spleen cells from the immunized animal with myeloma cells. Thefusion products are screened for those producing antibodies that bind tothe immunogen. The positive hybridomas clones are isolated, and themonoclonal antibodies are recovered from those clones.

Immunization regimens for production of both polyclonal and monoclonalantibodies are well known in the art. The immunogen may be injected byany of a number of routes, including subcutaneous, intravenous,intraperitoneal, intradermal, intramuscular, mucosal, or a combinationof these. The immunogen may be injected in soluble form, aggregate form,attached to a physical carrier, or mixed with an adjuvant, using methodsand materials well known in the art. The antisera and antibodies may bepurified using column chromatography methods well known to those ofskill in the art.

The antibodies may also be used as probes for identifying clones inexpression libraries that have or may have inserts encoding one or morePMP polypeptides or fragments thereof. The antibodies, PMP polypeptidesor PMP-derived polypeptides may also be used in immunoassays (e.g.,ELISA, RIA, Western Blots) to specifically detect and/or quantitateChlamydia or anti-Chlamydia antibody in biological specimens. Anti-PMPantibodies of the invention specifically bind PMP polypeptide fromChlamydia trachomatis, Chlamydia pneumoniae, Chlamydia pecorum, and/orChlamydia psittaci. Thus anti-PMP antibodies can be used to diagnoseChlamydia infections.

The antibodies of the invention, including but not limited to those thatare cytotoxic, cytostatic, or neutralizing, may also be used in passiveimmunization to prevent or attenuate Chlamydia infections of animals,including humans. As used herein, a cytotoxic antibody is one thatenhances opsonization and/or complement killing of the bacterium boundby the antibody. As used herein, neutralizing antibody is one thatreduces the infectivity of the Chlamydia and/or blocks binding ofChlamydia to a target cell. An effective concentration of polyclonal ormonoclonal antibodies raised against the immunogens of the invention maybe administered to a host to achieve such effects. The exactconcentration of the antibodies administered will vary according to eachspecific antibody preparation, but may be determined using standardtechniques well known to those of ordinary skill in the art.Administration of the antibodies may be accomplished using a variety oftechniques, including, but not limited to those described in Section 5.7for the delivery of vaccines.

Another aspect of the invention is directed to antisera raised againstan antigenic or immunogenic composition of the invention, and antibodiespresent in the antisera that specifically bind a PMP protein or afragment or analogue thereof.

Preferably, the antibodies bind a PMP polypeptide having the amino acidsequence selected from the group consisting of SEQ ID NOs.:2, 4-34 and73 and a PMP-derived polypeptide. Also included are monoclonalantibodies that specifically bind a PMP polypeptide having the aminoacid sequence selected from the group consisting of SEQ ID NOs.:2, 4-34,and 73 and a PMP-derived polypeptide. The term “antibodies” is intendedto include all forms, such as but not limited to polyclonal, monoclonal,purified IgG, IgM, or IgA antibodies and fragments thereof, includingbut not limited to antigen binding fragments such as Fv, single chain Fv(scFv), F(ab.)₂, Fab, and F(ab)′ fragments (Harlow and Lane, 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press);single chain antibodies (U.S. Pat. No. 4,946,778) and complementarydetermining regions (CDR), (see Verhoeyen and Winter, in MolecularImmunology 2ed., by B. D. Hames and D. M. Glover, IRL Press, OxfordUniversity Press, 1996, at pp. 283-325), etc.

A further aspect of the invention are chimeric or humanized antibodies(Morrison et al., 1984, Proc. Nat'l Acad. Sci. USA 81:6851; Reichmann etal. Nature 332:323: U.S. Pat. Nos. 5,225,539; 5,585,089; and 5,530,101;Neuberger et al., 1984, Nature 81:6851 Riechmann et al., 1988, Nature332:323; U.S. Pat. Nos. 5,225,539; 5,585,089; and 5,530,101) in whichone or more of the antigen binding regions of the anti-PMP antibody isintroduced into the framework region of a heterologous (e.g., human)antibody. The chimeric or humanized antibodies of the invention are lessantigenic in humans than non-human antibodies but have the desiredantigen binding and other activities, including but not limited toneutralizing activity, cytotoxic activity, opsonizing activity orprotective activity.

In a preferred embodiment, the antibodies of the invention are humanantibodies. Human antibodies may be isolated, for example, from humanimmunoglobulin libraries (see, e.g., PCT publications WO 98/46645, WO98/50433, WO 98/24893, WO 98/16054, WO 96/34096, WO 96/33735, and WO91/10741) by, preferably, phage display techniques (see, e.g., Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,616,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety. Human antibodies may also be generated fromanimals transgenic for one or more human immunoglobulin and that do notexpress endogenous immunoglobulins, see, e.g., U.S. Pat. No. 5,939,598by Kucherlapati et al.

A further aspect of the invention is T-cells specific for Chlamydia,Chlamydial EB, RBs, Chlamydia infected cells or antigen presenting cellsdisplaying Chlamydial antigens. T-cell preparations enriched for T-cellsspecific for PMP or PMP-derived polypeptides can be produced or isolatedby methods known in the art (See section 5.8).

5.5. Nucleic Acids Encoding the PMP Polypeptide and PMP DerivedPolypeptides

The isolated nucleic acids of the present invention, including DNA andRNA, comprising a sequence encoding the PMP protein or PMP-derivedpolypeptide thereof, may be synthesized using methods known in the art,such as using conventional chemical approaches or polymerase chainreaction (PCR) amplification using convenient pairs of oligonucleotideprimers and ligase chain reaction using a battery of contiguousoligonucleotides. The sequences also allow for the identification andcloning of the PMP protein gene from any species or serovar ofChlamydia, for instance for screening Chlamydial genomic libraries orexpression libraries as described below.

In a particular embodiment, the PMP polypeptide comprises an amino acidsequence of either SEQ ID NO.:2,4 or 73 and the nucleic acids comprisenucleotide sequences encoding said amino acid sequences. Particularlypreferred fragments of PMP have 8 or more amino acids of the amino acidsequences of SEQ ID NOs.:2, 4 or 73 or sequences substantiallyhomologous thereto, and the invention encompasses nucleic acidscomprising nucleotide sequences encoding said amino acid sequences. Inanother particular embodiment, the PMP polypeptide is encoded by thenucleotide sequence of SEQ ID NOs.:1, 3 or 72, with particularlypreferred fragments having a nucleotide sequence of NOs.:36-65, orsequences substantially homologous thereto.

The term “isolated nucleic acid” or “isolated nucleic acid molecule” isdefined as a nucleic acid molecule removed from the environment in whichit naturally occurs. For example, a naturally-occurring DNA moleculepresent in the genome of a living bacteria or as part of gene bank isnot isolated, but the same molecule separated from the remaining part ofthe bacterial genome, as a result of, e.g., a cloning event(amplification) is isolated. Typically, an isolated DNA molecule is freefrom DNA regions (e.g., coding regions) with which it is immediatelycontiguous at the 5′ or 3′ end, in the naturally occurring genome. Suchisolated nucleic acids or nucleic acid molecules could be part of avector or a composition and still be isolated in that such a vector orcomposition is not part of its natural environment. However, “isolatednucleic acid” or “isolated nucleic acid molecule” does not include anucleic acid that is part of a recombinant library.

Nucleic acids of the present invention can be single or double stranded.The invention also provides nucleic acids hybridizable to orcomplementary to SEQ ID NO.:1, 3 or 72 or fragments thereof, as well aspolypeptides encoded by these nucleic acids. In specific aspects,nucleic acids are provided which comprise a sequence fully complementaryto or complementary to at least 10, 15, 25, 50, 100, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100,1200, 1300, 1400 or 1500 contiguous nucleotides of a nucleic acidencoding a PMP polypeptide or an PMP-derived polypeptide. In a specificembodiment, a nucleic acid which is hybridizable to a nucleic acidencoding a PMP polypeptide (e.g., having a nucleotide sequence of SEQ IDNO.:1, 3 or 72 or to a nucleic acid encoding a PMP-derived polypeptide,under conditions of low, moderate or high stringency is provided). Alsoprovided are fragments of nucleic acids encoding a PMP polypeptide orPMP-derived polypeptide of the invention (or complements thereof) wheresuch fragments are at least 10, 15, 25, 50, 100, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100,1200, 1300, 1400 or 1500 nucleotides and in certain embodiments no morethan 50, 75, 100, 150, 200, 250, 300, 500, 600, 800, 1000, 1500, 2000,2500 or 3000 nucleotides in length.

By way of example and not limitation, procedures using such conditionsof low stringency are as follows (see also Shilo and Weinberg, 1981,Proc. Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA arepretreated for 6 h at 40° C. in a solution containing 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations arecarried out in the same solution with the following modifications: 0.02%PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol)dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 h at 40° C., and thenwashed for 1.5 h at 55° C. in a solution containing 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and re-exposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations).

In another specific embodiment, a nucleic acid which is hybridizable toa nucleic acid encoding a PMP polypeptide or a PMP-derived polypeptideunder conditions of high stringency is provided. By way of example andnot limitation, procedures using such conditions of high stringency areas follows: Prehybridization of filters containing DNA is carried outfor 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at65° C. in prehybridization mixture containing 100 μg/ml denatured salmonsperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters isdone at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01%Ficoll, and 0.01% BSA. This is followed by awash in 0.1×SSC at 50° C.for 45 min before autoradiography. Other conditions of high stringencywhich may be used are well known in the art.

In another specific embodiment, a nucleic acid which is hybridizable toa nucleic acid encoding a PMP polypeptide or a PMP-derived polypeptideunder conditions of moderate stringency is provided.

Various other stringency conditions which promote nucleic acidhybridization can be used. For example, hybridization in 6×SSC at about45° C., followed by washing in 2×SSC at 50° C. may be used.Alternatively, the salt concentration in the wash step can range fromlow stringency of about 5×SSC at 50° C., to moderate stringency of about2×SSC at 50° C., to high stringency of about 0.2×SSC at 50° C. Inaddition, the temperature of the wash step can be increased from lowstringency conditions at room temperature, to moderately stringentconditions at about 42° C., to high stringency conditions at about 65°C. Other conditions include, but are not limited to, hybridizing at 68°C. in 0.5M NaHPO₄ (pH7.2)/1 mM EDTA/7% SDS, or hybridization in 50%formamide/0.25M NaHPO₄ (pH 7.2)/0.25 M NaCl/1 mM EDTA/7% SDS; followedby washing in 40 mM NaHPO₄ (pH 7.2)/1 mM EDTA/5% SDS at 42° C. or in 40mM NaHPO₄ (pH7.2)/1 mM EDTA/1% SDS at 50° C. Both temperature and saltmay be varied, or alternatively, one or the other variable may remainconstant while the other is changed.

Low, moderate and high stringency conditions are well known to those ofskill in the art, and will vary predictably depending on the basecomposition of the particular nucleic acid sequence and on the specificorganism from which the nucleic acid sequence is derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Press, N.Y., pp. 9.47-9.57; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y.

In the preparation of genomic libraries, DNA fragments are generated,some of which will encode parts or the whole of a Chlamydia PMPE or PMPIprotein. The DNA may be cleaved at specific sites using variousrestriction enzymes. Alternatively, one may use DNase in the presence ofmanganese to fragment the DNA, or the DNA can be physically sheared, asfor example, by sonication. The DNA fragments can then be separatedaccording to size by standard techniques, including but not limited to,agarose and polyacrylamide gel electrophoresis, column chromatographyand sucrose gradient centrifugation. The DNA fragments can then beinserted into suitable vectors, including but not limited to plasmids,cosmids, bacteriophages lambda or T₄, bacmids and yeast artificialchromosome (YAC). (See, for example, Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning:A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) Thegenomic library may be screened by nucleic acid hybridization to labeledprobe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness,1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). The genomic librariesmaybe screened with labeled degenerate oligonucleotide probescorresponding to the amino acid sequence of any peptide of PMP proteinusing optimal approaches well known in the art. Any probe usedpreferably is 15 nucleotides or longer.

The term “probe” as used herein refers to DNA (preferably singlestranded) or RNA molecules that hybridize under stringent conditions asdefined above, to nucleic acids having sequences homologous to SEQ IDNO.:1, SEQ ID NO.:3, or SEQ ID NO:72, or to a complementary oranti-sense sequence thereof. Generally, probes are significantly shorterthan full-length sequences shown in SEQ ID NOs.:1, 3 or 72.

For example, they can contain from about 5 to about 100 nucleotidespreferably from about 10 to about 80 nucleotides. In particular, probeshave sequences that are at least 75% preferably at lest 85%, and morepreferably 95%, homologous to a portion of a sequence of SEQ ID NOs.:1,3 or 72, or complementary to such sequences. Probes can contain modifiedbases such as inosine, methyl-5-deoxycytidine, deoxyuridine,dimethylamino-5-deoxyuridine, or diamino-2,6 purine.

Clones in libraries with insert DNA encoding a PMP protein or aPMP-derived polypeptides will hybridize to one or more of the degenerateoligonucleotide probes. Hybridization of such oligonucleotide probes togenomic libraries is carried out using methods known in the art. Forexample, hybridization with the two above-mentioned oligonucleotideprobes may be carried out in 2×SSC, 1.0% SDS at 50° C. and washed usingthe same conditions.

In yet another aspect, clones of nucleotide sequences encoding a part orthe entire PMP protein or PMP-derived polypeptide may also be obtainedby screening Chlamydia expression libraries. For example, Chlamydia DNAor Chlamydia cDNA generated from RNA is isolated and random fragmentsare prepared and ligated into an expression vector (e.g., abacteriophage, plasmid, phagemid or cosmid) such that the insertedsequence in the vector is capable of being expressed by the host cellinto which the vector is then introduced. Various screening assays canthen be used to select for the expressed PMP-protein or PMP-derivedpolypeptides. In one embodiment, the various anti-PMP antibodies of theinvention can be used to identify the desired clones using methods knownin the art. See, for example, Harlow and Lane, 1988, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., Appendix IV. Clones or plaques from the library arebrought into contact with the antibodies to identify those clones thatbind.

In an embodiment, colonies or plaques containing DNA that encodes a PMPprotein or PMP-derived polypeptide could be detected using DYNA Beadsaccording to Olsvick et al., 29th ICAAC, Houston, Tex. 1989,incorporated herein by reference. Anti-PMP antibodies are crosslinked toDYNA Beads M280, and these antibody-containing beads are used to adsorbto colonies or plaques expressing a PMP protein or a PMP derivedpolypeptide. Colonies or plaques expressing a PMP protein or a PMPderived polypeptide are identified as any of those that bind the beads.

Alternatively, the anti-PMP antibodies can be nonspecificallyimmobilized to a suitable support, such as silica or Celite™ resin. Thismaterial is used to adsorb to bacterial colonies expressing a PMPprotein or a PMP derived polypeptide as described in the precedingparagraph.

In another aspect, PCR amplification may be used to producesubstantially pure DNA encoding a part of or the whole of a PMP proteinfrom Chlamydia genomic DNA. Oligonucleotide primers, degenerate orotherwise, corresponding to known PMP protein sequences can be used asprimers.

In particular embodiments, an oligonucleotide encoding a portion of SEQID NO.:2, 4 or 73 may be used as the 5′ primer. For fragment examples, a5′ primer may be made from any one of the nucleotide sequences of SEQ IDNO.:66 or 69 or any portion thereof. For 3′ primers, a nucleotidesequence of SEQ ID NO.:67 or 70 or any portion thereof may be used.

As examples, an oligonucleotide encoding the N-terminal primer, andtogether with a 3′ reverse PCR oligonucleotide complementary to aninternal, dow stream protein coding sequence may be used to amplify anN-terminal-specific PMP DNA fragment. Alternatively, an oligonucleotideencoding an internal PMP coding sequence may be used as the 5′ forwardPCR primer together with a 3′ reverse PCR oligonucleotide complementaryto downstream, internal PMP protein coding sequences may be used to PCRamplify an internal PMP specific DNA fragment. Alternatively, theforward primer can be combined together with an oligonucleotidecomplementary to the C-terminal PMP coding region to PCR amplify the PMPORF. These PMP specific PCR products can be cloned into appropriateexpression vectors to direct the synthesis of all or part of the PMPpolypeptide as distinct proteins or fusion proteins. Alternatively,these PMP specific PCR products can be appropriately labeled and used ashybridization probes to identify all or part of the PMP gene fromgenomic DNA libraries.

PCR can be carried out, e.g., by use of a Perkin-Elmer thermal cyclerand Taq polymerase (Gene Amp™). One can choose to synthesize severaldifferent degenerate primers, for use in the PCR reactions. It is alsopossible to vary the stringency of hybridization conditions used inpriming the PCR reactions, to allow for greater or lesser degrees ofnucleotide sequence similarity between the degenerate primers and thecorresponding sequences in Chlamydia DNA. After successful amplificationof a segment of the sequence encoding a PMP protein, that segment may bemolecularly cloned and sequenced, and utilized as a probe to isolate acomplete genomic clone. This, in turn, will permit the determination ofthe gene's complete nucleotide sequence, the analysis of its expression,and the production of its protein product for functional analysis, asdescribed infra.

Once a PMP polypeptide coding sequence has been isolated from oneChlamydia species, strain, or cultivar, it is possible to use the sameapproach to isolate PMP polypeptide coding sequences from otherChlamydia species, strains and cultivars. It will be recognized by thoseskilled in the art that the DNA or RNA sequence encoding PMPpolypeptides (or fragments thereof) of the invention can be used toobtain other DNA or RNA sequences that hybridize with it underconditions of moderate to high stringency, using general techniquesknown in the art (see supra). Hybridization with a PMP sequence from oneChlamydia strain or cultivar under high stringency conditions willidentify the corresponding sequences from other strains and cultivars.High stringency conditions vary with probe length and base composition.The formulae for determining such conditions are well known in the art.See Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, NY, Chapter 11. As an example, high stringencyhybridization conditions as applied to probes of greater than 300 basesin length involve a final wash in 0.1×SSC/0.1% SDS at 68° C. for atleast 1 hour (Ausubel, et al., Eds., 1989, Current Protocols inMolecular Biology, Vol. I, Greene Publishing Associates, Inc and JohnWiley & Sons, Inc. New York, at page 2.10.2). See also, the descriptionof high stringency conditions, supra.

One skilled in the art would be able to identify complete clones of aPMP polypeptide coding sequence using approaches well known in the art.The extent of the PMP polypeptide coding sequence contained in anisolated clone may be ascertained by sequencing the cloned insert andcomparing the deduced size of the polypeptide encoded by the openreading frames (ORFs) with that of the PMP polypeptide and/or bycomparing the deduced amino acid sequence with that of known amino acidsequence of the purified PMP polypeptide. Where a partial clone of thePMP polypeptide coding sequence has been isolated, complete clones maybe isolated by using the insert of the partial clone as a hybridizationprobe. Alternatively, a complete PMP polypeptide coding sequence can bereconstructed from overlapping partial clones by splicing their clonedPMP inserts together or by using the sequence information in the partialclones to design primers for PCR to amplify the entire PMP coding regionfrom an appropriate source such as Chlamydia genomic DNA or cDNA.

Complete clones may be any that an ORF with a deduced amino acidsequence matching or homologous to that of the PMP polypeptide or, wherethe complete amino acid sequence of the latter is not available,matching or homologous to that of a peptide fragment of a PMPpolypeptide and having a molecular weight corresponding to that of thePMP polypeptide. Further, complete clones may be identified by theability of their inserts, when placed in an expression vector, toproduce a polypeptide that binds antibodies specific to theamino-terminus of the PMP polypeptide and antibodies specific to thecarboxyl-terminus of the PMP polypeptide.

Nucleic acids encoding PMP-derived polypeptides and PMP fusion proteinsmay be produced by methods well known in the art. In one aspect, nucleicacids encoding PMP-derived polypeptides can be derived from PMPpolypeptide coding sequences by recombinant DNA methods in view of theteachings disclosed herein. For example, the coding sequence of a PMPpolypeptide may be altered creating amino acid substitutions that willnot affect the immunogenicity of the PMP polypeptide or which mayimprove its immunogenicity, such as conservative or semi-conservativesubstitutions as described above. Various methods may be used, includingbut not limited to, oligonucleotide directed, site specific mutagenesis.This and other techniques known in the art may be used to create singleor multiple mutations, such as replacements, insertions, deletions, andtranspositions, for example, as described in Botstein and Shortle, 1985,Science 229:1193-1210.

In another preferred embodiment, the nucleic acid encoding a PMPE orPMPI protein is a synthetic nucleic acid in which the codons have beenoptimized for increased expression in the host cell in which it isproduced. The degeneracy of the genetic code permits variations of thenucleotide sequence, while still producing a polypeptide having theidentical amino acid sequence as the polypeptide encoded by the nativeDNA sequence. The frequency of individual synonymous codons for aminoacids varies widely from genome to genome among eucaryotes andprocaryotes. These differences in codon choice patterns appear tocontribute to the overall expression levels of individual genes bymodulating peptide elongation rates. For this reason it is desirable anduseful to design nucleic acid molecules intended for a particularexpression system where the codon frequencies reflect the tRNAfrequencies of the host cell or organism in which the protein isexpressed. Native codons are exchanged for codons of highly expressedgenes in the host cells. For instance, the nucleic acid molecule can beoptimized for expression of the encoded protein in bacterial cells(e.g., E. coli), yeast (e.g., Pichia), insect cells (e.g., Drosophila),or mammalian cells or animals (e.g., human, sheep, bovine or mouse cellsor animals).

Restriction enzyme sites critical for gene synthesis and DNAmanipulation are preserved or destroyed to facilitate nucleic acid andvector construction and expression of the encoded protein. Inconstructing the synthetic genes of the invention it may be desirable toavoid CpG sequences as these sequences may cause gene silencing. Thus,in a preferred embodiment the coding region of the synthetic nucleicacid molecule does not include the sequence “CG” or includes less than5, 8, 10, 12, 15, 20 or 30 occurrences of the sequence “CG.” The codonoptimized sequence is synthesized and assembled and inserted into anappropriate expression vector using conventional techniques well knownto those of skill in the art.

In a particularly preferred embodiment, a synthetic nucleic acidencoding a PMPE or PMPI protein comprises at least one codonsubstitution in which non-preferred or less preferred codon in thenatural gene encoding the protein has been replaced by a preferred codonencoding the same amino acid. For instance in humans the preferredcodons are: Ala (GCC); Arg (CGC); Asn (AAC); Asp (GAC); Cys (TGC); Gln(CAG); Gly (GGC); His (CAC); Ile (ATC); Leu (CTG); Lys (AAG); Pro (CCC);Phe (TTC); Ser (AGC); Thr (ACC); Tyr (TAC); and Val (GTG). Lesspreferred codons are: Gly (GGG); Ile (ATT); Leu (CTC); Ser (TCC); Val(GTC); and Arg (AGG). All codons which do not fit the description ofpreferred codons or less preferred codons are non-preferred codons. Ingeneral, the degree of preference of a particular codon is indicated bythe prevalence of the codon in highly expressed genes. Codon preferencefor highly expressed human genes are as indicated in Table 3. Forexample, “ATC” represents 77% of the Ile codons in highly expressedmammalian genes and is the preferred Ile codon; “ATT” represents 18% ofthe Ile codons in highly expressed mammalian genes and is the lesspreferred Ile codon. The sequence “ATA” represents only 5% of the Ilecodons in highly expressed human genes and is a non-preferred Ile codon.Replacing a codon with another codon that is more prevalent in highlyexpressed human genes will generally increase expression of the gene inmammalian cells. Accordingly, the invention includes replacing a lesspreferred codon with a preferred codon as well as replacing anon-preferred codon with a preferred or less preferred codon.

The synthetic nucleic acid is optimized for expression of the encodedprotein and at least one non-preferred or less preferred coding in anucleic acid molecule encoding the protein is replaced by a preferred ormore preferred codon encoding the same amino acid. The synthetic nucleicacid expresses the encoded protein at a level which is at least 110%,125%, 150%, 200%, 500% of that expressed by the starting nucleic acidmolecule (i.e., prior to optimization) in an in vitro cell culturesystem under identical conditions. In addition, preferably the syntheticnucleic acid molecule comprises fewer than 5, 8, 10, 12, 15, 20 or 30occurrences of the sequence CG. Preferably at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80% or 90% of the non-preferred codons and less preferredcodons in the nucleic acid molecule have been replaced by preferredcodons or more preferred codons.

In a particularly preferred embodiment, the nucleic acid has beenoptimized for expression of the encoded protein in human or mammaliancells or organisms. TABLE 3 Codon Frequency (Percentage) in highlyexpressed human genes Ala GC C 53 T 17 A 13 G 17 Arg CG C 37 T 7 A 6 G21 AG A 10 G 18 Asn AA C 78 T 22 Asp GA C 75 T 25 Leu CT C 26 T 5 A 3 G58 TT A 2 G 6 Lys AA A 18 G 82 Pro CC C 48 T 19 A 16 G 17 Phe TT C 80 T20 Cys TG C 68 T 32 Gln CA A 12 G 88 Glu GA A 25 G 75 Gly GG C 50 T 12 A14 G 24 His CA C 79 T 21 Ile AT C 77 T 18 A 5 Ser TC C 28 T 13 A 5 G 9AG C 34 T 10 Thr AC C 57 T 14 A 14 G 15 Tyr TA C 74 T 26 Val GT C 25 T 7A 5 G 64

Further, nucleic acids containing PMP polypeptide coding sequences maybe truncated by restriction enzyme or exonuclease digestions.Heterologous coding sequences may be added to the PMP polypeptide codingsequence by ligation or PCR amplification Moreover, DNA encoding thewhole or a part of PMP-derived polypeptide may be synthesized chemicallyor using PCR amplification based on the known or deduced amino acidsequence of the PMP polypeptide and any desired alterations to thatsequence.

The identified and isolated DNA containing the PMP polypeptide orPMP-derived polypeptide coding sequence can be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. The term “host” as used herein and in the claimsrefers to either in vivo in an animal or in vitro in mammalian cellcultures.

Possible vectors include, but are not limited to, plasmids and modifiedviruses, but the vector system must be compatible with the host cellused. Such vectors include, but are not limited to, bacteriophage suchas lambda derivatives, or plasmids such as pET, pBAD, pTrcHis, pBR322 orpUC plasmid derivatives. The insertion into a cloning vector can, forexample, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved DNA may be modified byhomopolymeric tailing. Recombinant molecules can be introduced into hostcells via transformation, transfection, infection, electroporation,etc., so that many copies of the gene sequence are generated.

In an alternative method, the desired DNA containing a PMP polypeptideor PMP-derived polypeptide coding sequence may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired sequence, for example, by sizefractionation, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that contain a PMP polypeptide or PMP-derived polypeptidecoding sequence enables generation of multiple copies of such codingsequence. Thus, the coding sequence may be obtained in large quantitiesby growing transformants, isolating the recombinant DNA molecules fromthe transformants and, when necessary, retrieving the inserted codingsequence from the isolated recombinant DNA.

The nucleotide sequences encoding the PMP polypeptides of the presentinvention are useful for their ability to selectively form duplexmolecules with complementary stretches of other protein genes. Dependingon the application, a variety of hybridization conditions may beemployed to achieve hybridization with varying sequence identities. Inspecific aspects, nucleic acids are provided which comprise a sequencecomplementary to at least 10, 15, 25, 50, 100, 200 or 250 nucleotides ofthe PMP protein coding nucleic acid molecule. In specific embodiments,nucleic acids which hybridize to a PMP protein nucleic acid (e.g.,having a nucleotide sequence of SEQ ID NO.:1, 3 or 72) under annealingconditions are provided.

For a high degree of selectivity, relatively stringent conditions areused to form the duplexes, such as, by way of example and notlimitation, low salt and/or high temperature conditions, such asprovided by 0.02 M to 0.15 M NaCl at temperatures of between about 50°C. to 70° C. For some applications, less stringent hybridizationconditions are required, by way of example and not limitation, such as0.15 M to 0.9 M salt, at temperatures ranging from between about 20° C.to 55° C. Hybridization conditions can also be rendered more stringentby the addition of increasing amounts of formamide, to destabilize thehybrid duplex. Thus, particular hybridization conditions can be readilymanipulated, and will generally be a method of choice depending on thedesired results.

5.6. Recombinant Production of PMP Polypeptide and PMP-DerivedPolypeptides

In accordance with this invention, it is preferred to make the Chlamydiaprotein of the present invention by recombinant methods, particularlywhen the naturally occurring protein as isolated from a culture of aspecies of Chlamydia may include trace amounts of toxic materials orother contaminants. This problem can be avoided by using proteinrecombinantly produced in heterologous systems which can be isolatedfrom the host in a manner to minimize contaminants in the isolatedmaterial. In this case, the PMP proteins are produced by an appropriatehost cell that has been transformed by a DNA molecule that codes for thepolypeptide.

The nucleic acids encoding the PMP polypeptides or PMP-derivedpolypeptides of the invention can be inserted into an appropriateexpression vector, i.e., a vector that contains the necessary elementsfor the transcription and translation of the inserted polypeptide-codingsequence. The nucleotide sequences encoding the PMP polypeptides orPMP-derived polypeptides are inserted into the vectors in a manner suchthat they will be expressed under appropriate conditions (e.g., inproper orientation and correct reading frame). The recombinantexpression vector also comprises an “expression means”. The term“expression means” refers to elements of a vector which are necessaryfor transcription and translation of the nucleic acid encoding theprotein, including but not limited to promoter/enhancer elements, areplication site, an RNA polymerase binding sequence, a ribosomalbinding sequence, sequences which are capable of providing phenotypeselection (e.g., ampicillin or tetracycline resistance), peptide tagsthat permit isolation of the expressed protein, signal sequences thatdirect secretion of the expressed protein and replicon and controlsequences that can be used to transform host cells. The expression meansis operatively coupled to the nucleic acid molecule encoding the PMPprotein by linking the inserted nucleic acid molecule into theexpression vector.

Promoter/enhancer elements which may be used to control expression ofinserted sequences include, but are not limited to the SV40 earlypromoter region (Bernoist and Chambon, 1981, Nature 290:304-310), thepromoter contained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinasepromoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42) for expression in animalcells; the promoters of lactamase (Villa-Kamaroff et al., 1978, Proc.Natl. Acad. Sci. U.S.A. 75:3727-3731), tac (DeBoer et al., 1983, Proc.Natl. Acad. Sci. U.S.A. 80:21-25), or trc for expression in bacterialcells (see also “Useful proteins from recombinant bacteria” inScientific American, 1980, 242:74-94); the nopaline synthetase promoterregion or the cauliflower mosaic virus 35S RNA promoter (Gardner et al.,1981, Nucl. Acids Res. 9:2871), and the promoter of the photosyntheticenzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984,Nature 310:115-120) for expression in plant cells; Gal4 promoter, theADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter for expression in yeast or otherfungi.

Depending on the host-vector system utilized, any one of a number ofsuitable transcription and translation elements may be used. In apreferred embodiment, a chimeric protein comprising a PMP protein orPMP-derived polypeptide sequence and a pre and/or pro sequence of thehost cell is expressed. In other preferred embodiments, a chimericprotein comprising a PMP protein or PMP derived polypeptide sequencefused with, for example, an affinity purification peptide, including butnot limited to, maltose binding protein, glutathione-S-transferase,thioredoxin or histidine tag, is expressed. In further preferredembodiments, a chimeric protein comprising a PMP protein or PMP-derivedpolypeptide sequence and a useful immunogenic peptide or protein isexpressed.

Any method known in the art for inserting DNA fragments into a vectormay be used to construct expression vectors containing a PMP orPMP-derived polypeptide encoding nucleic acid molecule consisting ofappropriate transcriptional/translational control signals and thepolypeptide coding sequences. These methods may include in vitrorecombinant DNA and synthetic techniques and in vivo recombinantation(genetic recombination).

Methods of introducing exogenous DNA into yeast hosts include either thetransformation of spheroplasts or of intact yeast cells treated withalkali cations. Transformation procedures usually vary with the yeastspecies to be transformed. See e.g., Kurtz et al. (1986) Mol. Cell.Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; forCandida, Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkampet al. (1986) Mol. Gen. Genet. 202:302; for Hansenula; Das et al. (1984)J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol.154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; forKluyveromyces; Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et al.(1985) J. Basic Microbiol. 25:141; U.S. Pat. No. 4,837,148 and U.S. Pat.No. 4,929,555; for Pichia; Hinnen et al. (1978) Proc. Natl. Acad. Sci.USA 75;1929; Ito et al. (1983) J. Bacteriol. 153:163; for Saccharomyces;Beach et al. (1981) Nature 300:706; for Schizosaccharomyces; Davidow etal. (1985) Curr. Genet. 10:39.

Expression vectors containing PMP polypeptide or PMP-derived polypeptidecoding sequences can be identified by three general approaches: (a)nucleic acid hybridization; (b) presence or absence of “marker” genefunctions; and (c) expression of inserted sequences. In the firstapproach, the presence of a foreign gene inserted into an expressionvector can be detected by nucleic acid hybridization using probescomprising sequences that are homologous to the inserted PMP polypeptideor PMP-derived polypeptide coding sequence. In the second approach, therecombinant vector/host system can be identified and selected based uponthe presence or absence of certain “marker” gene functions (e.g.,thymidine kinase activity, resistance to antibiotics, transformationphenotype, occlusion body formation in baculovirus, etc.) caused by theinsertion of foreign genes into the vector.

For example, E. coli may be transformed using pBR322 which containsgenes for ampicillin and tetracycline resistance. If the PMP polypeptideor PMP-derived polypeptide coding sequence is inserted within the markergene sequence of the vector, recombinants containing the insert can beidentified by the absence of the marker gene function. In the thirdapproach, recombinant expression vectors can be identified by assayingthe foreign gene product expressed by the recombinant. Such assays canbe based, for example, on the physical or functional properties of PMPpolypeptide or PMP-derived polypeptide in vitro assay systems, e.g.,binding of a His tag engineered into the expressed protein to a column,binding to a PMP ligand or receptor or binding with anti-PMP antibodiesof the invention.

Commercially available vectors for expressing heterologous proteins inbacterial hosts include but are not limited to pZERO, pTrc99A, pUC19,pUC18, pKK223-3, pEX1, pCAL, pET, pSPUTK, pTrxFus, pFastBac, pThioHis,pTrcHis, pTrcHis2, and pLEx. For example, the phage in lambda GEM™-11may be utilized in making recombinant phage vectors which can be used totransform host cells, such as E. coli LE392. In a preferred embodiment,the vector is pQE30 or pBAD/ThioE, which can be used transform hostcells, such as E. coli.

Expression and transformation vectors for transformation into many yeaststrains are available. For example, expression vectors have beendeveloped for, the following yeasts: Candida albicans, Kurtz, et al.(1986) Mol. Cell. Biol. 6:142; Candida maltosa, Kunze, et al. (1985) J.Basic Microbiol. 25:141; Hansenula polymorpha, Gleeson, et al. (1986) J.Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet.202:302; Kluyveromyces fragilis, Das, et al. (1984) J. Bacteriol.158:1165; Kluyveromyces lactis, De Louvencourt et al. (1983) J.Bacteriol. 154:737; Van den Berg, et al. (1990) Bio/Technology 8:135;Pichia quillerimondii, Kunze et al. (1985) J. Basic Microbiol. 25:141;Pichia pastoris, Cregg, et al. (1985) Mol. Cell. Biol. 5:3376, U.S. Pat.No. 4,837,148 and U.S. Pat. No. 4,929,555; Saccharomyces cerevisiae,Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929, Ito et al.(1983) J. Bacteriol. 153:163; Schizosaccharomyces pombe, Beach et al.(1981) Nature 300:706; and Yarrowia lipolytica, Davidow, et al. (1985)Curr. Genet. 10:380-471, Gaillardin, et al. (1985) Curr. Genet. 10:49.

A variety of host-vector systems may be utilized to express thepolypeptide-coding sequence. These include but are not limited tomammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors, orbacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA,plant cells or transgenic plants.

Hosts that are appropriate for expression of nucleic acid molecules ofthe present invention, fragments, analogues or variants thereof, mayinclude E. coli, Bacillus species, Haemophilus, fungi, yeast, such asSaccharomyces, Pichia, Bordetella, or Candida, or the baculovirusexpression system. Preferably, the host cell is a yeast or bacterium. Inone embodiment, the host cell is an E. coli cell which has beengenetically engineered to express epitopes of C. trachomatis LPS (see,e.g., U.S. Pat. No. 5,075,228, which is hereby incorporated by referencein its entirety). In a preferred embodiment, the PMP protein orPMP-derived protein is expressed in a heterologous, recombinant bacteriawhich has been engineered to express lpxA and/or Kdo transferase of C.trachomatis (for nucleotide and amino acid sequences see Genbankaccession numbers AEOO1324 and AE001294, which are hereby incorporatedby reference in their entirety) and is defective in its own lpxA or Kdotransferase gene. In other embodiments, the host also expresses one ormore Chlamydia, preferably Chlamydia trachomatis, proteins, preferablyan outer membrane protein, most preferably HMW protein, MOMP, PMPH orHtrA, or a fragment thereof.

Particularly desirable hosts for expression in this regard include Grampositive bacteria which do not have LPS and are, therefore endotoxinfree. Most preferably the bacterium is E. coli, B. subtilis orSalmonella.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered PMP polypeptide or PMP-derivedpolypeptide may be controlled. Furthermore, different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to ensure the desiredmodification and processing of the foreign protein expressed.

Once a suitable host system and growth conditions are established,recombinant expression vectors can be propagated and prepared inquantity. Upon expression, a recombinant polypeptide of the invention isproduced and can be recovered in a substantially purified from the cellpaste, the cell extract or from the supernatant after centrifugation ofthe recombinant cell culture using techniques well known in the art.

For instance, the recombinant polypeptide can be purified byantibody-based affinity purification, preparative gel electrophoresis,or affinity purification using tags (e.g., 6× histidine tag) included inthe recombinant polypeptide. (See, Section 5.3 supra).

5.7. Compositions

The present invention also provides therapeutic and prophylacticcompositions, which may be antigenic compositions, and preferablyimmunogenic compositions, including vaccines, for use in the treatmentor prevention of Chlamydia infections of animals, including mammals andbirds, and more specifically rodents and primates, including humans.Preferred immunogenic compositions include vaccines for use in humans.The antigenic, preferably immunogenic, compositions of the presentinvention can be prepared by techniques known to those skilled in theart and comprise, for example, an immunologically effective amount ofany of the PMP immunogens disclosed in Sections 5.1. or 5.2, optionallyin combination with or fused to or conjugated to one or more otherimmunogens, including lipids, phospholipids, carbohydrates,lipopolysaccharides, inactivated or attenuated whole organisms and otherproteins, of Chlamydia origin or other bacterial origin, apharmaceutically acceptable carrier, optionally an appropriate adjuvant,and optionally other materials traditionally found in vaccines. In oneembodiment, the invention provides a cocktail vaccine comprising severalimmunogens, which has the advantage that immunity against one or severalstrains of a single pathogen or one or several pathogens can be obtainedby a single administration. Examples of other immunogens include, butare not limited to, those used in the known DPT vaccines, HMW protein ofC. trachomatis or fragments thereof, MOMP of C. trachomatis or fragmentsthereof, or PMPH or HtrA of C. trachomatis or fragments thereof(preferably epitope containing fragments), entire organisms or subunitstherefrom of Chlamydia, Neisseria, HIV, Haemophilus influenzae,Moraxella catarrhalis, Human papilloma virus, Herpes simplex virus,Haemophilus ducreyi, Treponema pallidium, Candida albicans andStreptococcus pneumoniae, etc. Preferred are compositions comprising oneor more fragments of C. trachomatis MOMP which comprise or consist ofresidues 273-333, 64-85, 139-160, 224-237, 288-317, 1-200, 64-350,160-350, 240-333 or 80-105. The compositions may optionally comprise HMWprotein or comprise an amino-terminal fragment of HMW protein, i.e., afragment comprising or consisting of residues 1-100, 1-200, 1-300,1-400, or 1-500 of the mature HMW protein. The compositions mayoptionally comprise Chlamydia trachomatis LPS or LPS from a recombinantbacteria which has been engineered to express lpxA or Kdo transferase ofC. trachomatis and which is defective in its own lpxA or Kdo transferasegene. For instance, LPS from an E. coli mutant defective in its lpxAtransfected with nucleic acid encoding lpxA protein of C. trachomatiscan be isolated and purified according to techniques well known in theart (Sweet et al., J. Biol. Chem. 276:19565, 2001 or Rund et al. J.Biol. Chem. 274:16819, 1999, both of which are incorporated byreferenced in their entireties).

In specific embodiments, the pharmaceutical or vaccine compositioncomprises a PMPE or PMPI polypeptide or PMPE-derived polypeptide orPMPI-derived polypeptide and an HMW protein, or fragment thereof(preferably an at least 5, 8, 10, 20, 40, 50, 60, 80, 100, 150, 200,300, 400 or 500 amino acid fragment and preferably an epitope containingfragment thereof). In other specific embodiments, the compositioncomprises a PMPE or PMPI polypeptide or PMPE-derived polypeptide orPMPI-derived polypeptide and a MOMP, or fragment thereof (preferably anat least 5, 8, 10, 20, 40, 50, 60, 80, 100, 150, 200, 300, 400 or 500amino acid fragment and preferably an epitope containing fragmentthereof).

The term “immunogenic amount” is used herein to mean an amountsufficient to induce an immune response to produce antibodies, T-cells,and/or cytokines and other cellular immune response components.Preferably, the immunogenic composition is one that elicits an immuneresponse sufficient to prevent Chlamydia infections or to attenuate theseverity of any preexisting or subsequent Chlamydia infection. Animmunogenic amount of the immunogen to be used in the vaccine isdetermined by means known in the art in view of the teachings herein.The exact concentration will depend upon the specific immunogen to beadministered, but can be determined by using standard techniques wellknown to those skilled in the art for assaying the development of animmune response.

The vaccine compositions of the invention elicit an immune response in asubject. Compositions which induce antibodies, including anti-PMPprotein antibodies and antibodies that are opsonizing or bactericidalare one aspect of the invention. In preferred non-limiting, embodimentsof the invention, an effective amount of a composition of the inventionproduces an elevation of antibody titer to at least three times theantibody titer prior to administration. In a preferred, specific,non-limiting embodiment of the invention, approximately 0.01 to 2000 μgand, preferably, 0.1 to 500 μg, most preferably 50 to 250 μg of the PMPprotein or PMP-derived protein is administered is to a host.Compositions which induce T-cell responses which are bactericidal orreactive with host cells infected with Chlamydia are also an aspect ofthe invention. Preferred are compositions additionally comprising anadjuvant.

The combined immunogen and carrier or diluent may be an aqueoussolution, emulsion or suspension or may be a dried preparation.Appropriate carriers are known to those skilled in the art and includestabilizers, diluents, and buffers. Suitable stabilizers includecarbohydrates, such as sorbitol, lactose, mannitol, starch, sucrose,dextran, and glucose, and proteins, such as albumin or casein. Suitablediluents include saline, Hanks Balanced Salts, and Ringers solution.Suitable buffers include an alkali metal phosphate, an alkali metalcarbonate, or an alkaline earth metal carbonate. In preferredembodiments, the composition of the invention is formulated foradministration to humans.

The pharmaceutical and immunogenic compositions, including vaccines, ofthe invention are prepared by techniques known to those skilled in theart, given the teachings contained herein. Generally, an immunogen ismixed with the carrier to form a solution, suspension, or emulsion. Oneor more of the additives discussed herein may be added in the carrier ormay be added subsequently. The vaccine preparations may be desiccated orlyophilized, for example, by freeze drying or spray drying for storageor formulations purposes. They may be subsequently reconstituted intoliquid vaccines by the addition of an appropriate liquid carrier oradministered in dry formulation using methods known to those skilled inthe art, particularly in capsules or tablet forms.

An effective amount of the antigenic, immunogenic, pharmaceutical,including, but not limited to vaccine, composition of the inventionshould be administered, in which “effective amount” is defined as anamount that is sufficient to produce a desired prophylactic, therapeuticor ameliorative response in a subject, including but not limited to animmune response. The amount needed will vary depending upon theimmunogenicity of the PMP protein, PMP-derived polypeptide or nucleicacid used, and the species and weight of the subject to be administered,but maybe ascertained using standard techniques.

Immunogenic, antigenic, pharmaceutical and vaccine compositions mayfurther contain one or more auxiliary substance, such as wetting oremulsifying agents, pH buffering agents, or adjuvants to enhance theeffectiveness thereof. Immunogenic, antigenic, pharmaceutical andvaccine compositions may be administered to birds, humans or othermammals, including ruminants, rodents or primates, by a variety ofadministration routes, including parenterally, intradermally,intraperitoneally, subcutaneously or intramuscularly.

Alternatively, the immunogenic, antigenic, pharmaceutical and vaccinecompositions formed according to the present invention, may beformulated and delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic, antigenic, pharmaceutical andvaccine compositions may be administered to mucosal surfaces by, forexample, the nasal, oral (intragastric), ocular, bronchiolar,intravaginal or intrarectal routes. Alternatively, other modes ofadministration including suppositories and oral formulations may bedesirable. For suppositories, binders and carriers may include, forexample, polyalkalene glycols or triglycerides. Oral formulations mayinclude normally employed incipients such as, for example,pharmaceutical grades of saccharine, cellulose and magnesium carbonate.These compositions can take the form of microspheres, solutions,suspensions, tablets, pills, capsules, sustained release formulations orpowders and contain about 0.001 to 95% of the PMP protein. Preferreddosage forms contain 50 μg to 250 μg of the PMP protein. Theimmunogenic, antigenic, pharmaceutical and vaccine compositions areadministered in a manner compatible with the dosage formulation, and insuch amount as will be therapeutically effective, protective orimmunogenic. Preferred are compositions additionally comprising anadjuvant.

Further, the immunogenic, antigenic, pharmaceutical and vaccinecompositions may be used in combination with or conjugated to one ormore targeting molecules for delivery to specific cells of the immunesystem and/or mucosal surfaces. Some examples include but are notlimited to vitamin B12, bacterial toxins or fragments thereof,monoclonal antibodies and other specific targeting lipids, proteins,nucleic acids or carbohydrates.

Suitable regimes for initial administration and booster doses are alsovariable, but may include an initial administration followed bysubsequent administrations. The dose may also depend on the route(s) ofadministration and will vary according to the size of the host. Theconcentration of the PMP protein or PMP-derived polypeptide in anantigenic, immunogenic or pharmaceutical composition according to theinvention is in general about 0.001 to 95%, preferably 0.01 to 5%.

The antigenic, immunogenic or pharmaceutical preparations, includingvaccines, may comprise as the immunostimulating material a nucleic acidvector comprising at least a portion of the nucleic acid moleculeencoding the PMP protein or PMP-derived polypeptide.

A vaccine comprising nucleic acid molecules encoding one or more PMPpolypeptides, PMP-derived polypeptides or fusion proteins as describedherein, such that the polypeptide is generated in situ is provided. Insuch vaccines, the nucleic acid molecules may be present within any of avariety of delivery systems known to those skilled in the art, includingnucleic acid expression systems, bacterial and viral expression systems.Appropriate nucleic acid expression systems contain the necessarynucleotide sequences for expression in the patient such as suitablepromoter and terminating signals. In a preferred embodiment, the nucleicacid molecules may be introduced using a viral expression system (e.g.,vaccinia or other pox virus, alphavirus retrovirus or adenovirus) whichmay involve the use of non-pathogenic (defective) virus. Techniques forincorporating nucleic acid molecules into such expression systems arewell known to those of ordinary skill in the art. The nucleic acidmolecules may also be administered as “naked” plasmid vectors asdescribed, for example, in Ulmer et al. Science 259:1745-1749 (1992) andreviewed by Cohen, Science 259:1691-1692 (1993). Techniques forincorporating DNA into such vectors are well known to those of ordinaryskill in the art. A vector may additionally transfer or incorporate agene for a selectable marker (to aid in the identification or selectionof transduced cells) and/or a targeting moiety, such as a gene thatencodes a ligand for a receptor on a specific target cell, to render thevector target specific. Targeting may also be accomplished using anantibody, by methods know to those skilled in the art.

Nucleic acid molecules (DNA or RNA) of the invention can be administeredas vaccines for therapeutic or prophylactic purpose. Typically a DNAmolecule is placed under the control of a promoter suitable forexpression in a mammalian cell. The promoter can function ubiquitouslyor tissue-specifically. Examples of non-tissue specific promotersinclude the early cytomegalovirus (CMV) promoter (described in U.S. Pat.No. 4,168,062) and Rous Sarcoma virus promoter (described in Norton andCoffin, Molec. Cell Biol. 5:281 (1985)). The desmin promoter (Li et al.Gene 78:243 (1989); Li & Paulin, J. Biol Chem 266:6562 (1991); and Li &Paulin, J. Biol Chem 268:10401 (1993)) is tissue specific and drivesexpression in muscle cells. More generally, useful vectors are describedin, e.g., WO 94/21797 and Hartikka et al., Human Gene Therapy 7:1205(1996).

A composition of the invention can contain one or several nucleic acidmolecules of the invention. It can also contain at least one additionalnucleic acid molecule encoding another antigen or fragment derivative,including but not limited to, DPT vaccines, HMW protein of C.trachomatis or fragment thereof, MOMP of C. trachomatis or fragmentthereof, entire organisms or subunits therefrom of Chlamydia, Neisseria,HIV, Haemophilus influenzae, Moraxella catarrhalis, Human papillomavirus, Herpes simplex virus, Haemophilus ducreyi, Treponema pallidium,Candida albicans and Streptococcus pneumoniae, etc. A nucleic acidmolecule encoding a cytokine, such as interleukin-1 or interleukin-12can also be added to the composition so that the immune response isenhanced. DNA molecules of the invention and/or additional DNA moleculesmay be on different plasmids or vectors in the same composition or canbe carried in the same plasmid or vector.

Other formulations of nucleic acid molecules for therapeutic andprophylactic purposes include sterile saline or sterile buffered salinecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, silica microparticles, tungsten microparticles, goldmicroparticles, microspheres, beads and lipid based systems includingoil-in-water emulsions, micelles, mixed micelles and liposomes. Apreferred colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (i.e., an artificial vesicle). The uptake of nakednucleic acid molecules may be increased by incorporating the nucleicacid molecules into and/or onto biodegradable beads, which areefficiently transported into the cells. The preparation and use of suchsystems is well known in the art.

A nucleic acid molecule can be associated with agents that assist incellular uptake. It can be formulated with a chemical agent thatmodifies the cellular permeability, such as bupivacaine (see, e.g., WO94/16737).

Cationic lipids are also known in the art and are commonly used for DNAdelivery. Such lipids include LIPOFECTIN™, also known as DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP(1,2-bis(oleyloxy)-3-(trimethylammonio)propane, DDAB(dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyspermine) and cholesterol derivatives such as DC-Chol (3beta-(N-(N′,N′-dimethyl aminomethane)-carbamoyl)cholesterol. Adescription of these cationic lipids can be found in EP 187,702, WO90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S.Pat. No. 5,527,928. Cationic lipids for DNA delivery are preferably usedin association with a neutral lipid such as DOPE (dioleylphosphatidylethanolamine) as described in, e.g., WO 90/11092.

Other transfection facilitation compounds can be added to a formulationcontaining cationic liposomes. They include, e.g., spermine derivativesuseful for facilitating the transport of DNA through the nuclearmembrane (see, for example, WO 93/18759) and membrane-permeabilizingcompounds such as GALA, Gramicidine S and cationic bile salts (see, forexample, WO 93/19768).

The amount of nucleic acid molecule to be used in a vaccine recipientdepends, e.g., on the strength of the promoter used in the DNAconstruct, the immunogenicity of the expressed gene product, the mode ofadministration and type of formulation. In general, a therapeutically orprophylactically effective dose from about 1 μg to about 1 mg,preferably from about 10 μg to about 800 μg and more preferably fromabout 25 μg to about 250 μg can be administered to human adults. Theadministration can be achieved in a single dose or repeated atintervals.

The route of administration can be any conventional route used in thevaccine field. As general guidance, a nucleic acid molecule of theinvention can be administered via a mucosal surface, e.g., an ocular,intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinarytract surface; or via a parenteral route, e.g., by an intravenous,subcutaneous, intraperitoneal, intradermal, intra-epidermal orintramuscular route. The choice of administration will depend on theformulation that is selected. For instance a nucleic acid moleculeformulated in association with bupivacaine is advantageouslyadministered into muscles.

Recombinant bacterial vaccines genetically engineered for recombinantexpression of nucleic acid molecules encoding PMP or PMP-derivedpolypeptides include Shigella, Salmonella, Vibrio cholerae, andLactobacillus. Recombinant BCG and Streptococcus expressing PMP orPMP-derived polypeptides can also be used for prevention or treatment ofChlamydia infections.

Non-toxicogenic Vibrio cholerae mutant strains that are useful as a liveoral vaccine are described in Mekalanos et al. Nature 306:551 (1983) andU.S. Pat. No. 4,882,278. An effective vaccine dose of a Vibrio choleraestrain capable of expressing a polypeptide or polypeptide derivativeencoded by a DNA molecule of the invention can be administered.Preferred routes of administration include all mucosal routes, mostpreferably intranasally or orally.

Attenuated Salmonella typhimurium strains, genetically engineered forrecombinant expression of heterologous antigens or not and their use asoral vaccines are described in Nakayama et al. Bio/Technology 6:693(1988) and WO 92/11361. Preferred routes of administration include allmucosal routes, most preferably intranasally or orally.

Other bacterial strains useful as vaccine vectors are described in Highet al., EMBO 11:1991(1992); Sizemore et al., Science 270:299 (1995)(Shigella flexneri); Medaglini et al., Proc Natl. Acad. Sci. US 92:6868(1995) (Streptococcus gordonii); and Flynn, Cell Mol. Biol. 40:31(1994); WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 02/21376(Bacille Calmette Guerin).

In genetically engineered recombinant bacterial vectors, nucleic acidmolecule(s) of the invention can be inserted into the bacterial genome,carried on a plasmid, or can remain in a free state.

When used as vaccine agents, recombinant bacterial or viral vaccines,nucleic acid molecules and polypeptides of the invention can be usedsequentially or concomitantly as part of a multistep immunizationprocess. For example, a mammal or bird can be initially primed with avaccine vector of the invention such as pox virus or adenovirus, e.g.,via the parenteral route or mucosally and then boosted several time withthe a polypeptide e.g., via the mucosal route. In another example, amammal can be vaccinated with polypeptide via the mucosal route and atthe same time or shortly thereafter, with a nucleic acid molecule viaintramuscular route.

An adjuvant can also be added to a composition containing a PMP vaccine.To efficiently induce humoral immune responses (HIR) and cell-mediatedimmunity (CMI), immunogens are typically emulsified in adjuvants.Immunogenicity can be significantly improved if the immunogen isco-administered with an adjuvant. Adjuvants may act by retaining theimmunogen locally near the site of administration to produce a depoteffect facilitating a slow, sustained release of antigen to cells of theimmune system. Adjuvants can also attract cells of the immune system toan immunogen depot and stimulate such cells to elicit immune responses.

Many adjuvants are toxic, inducing granulomas, acute and chronicinflammations (Freund's complete adjuvant, FCA), cytolysis (saponins andPluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPSand MDP). Although FCA is an excellent adjuvant and widely used inresearch, it is not licensed for use in human or veterinary vaccinesbecause of its toxicity.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccharides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are typically non-covalently linked toantigens and are formulated to enhance the host immune responses. Thus,adjuvants have been identified that enhance the immune response toantigens delivered parenterally. Aluminum hydroxide, aluminum oxide, andaluminum phosphate (collectively commonly referred to as alum) areroutinely used as adjuvants in human and veterinary vaccines. Theefficacy of alum in increasing antibody responses to diphtheria andtetanus toxoids is well established and a HBsAg vaccine has beenadjuvanted with alum.

Other extrinsic adjuvants may include chemokines, cytokines (e.g.,IL-2), saponins complexed to membrane protein antigens (immunestimulating complexes), pluronic polymers with mineral oil, killedmycobacteria in mineral oil, Freund's complete adjuvant, bacterialproducts, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS),as well as lipid A, and liposomes.

U.S. Pat. No. 6,019,982, incorporated herein by reference, describesmutated forms of heat labile toxin of enterotoxigenic E. coli (“mLT”).U.S. Pat. No. 5,057,540, incorporated herein by reference, describes theadjuvant, QS21, an HPLC purified non-toxic fraction of a saponin fromthe bark of the South American tree Quiliaja saponaria molina. 3D-MPL isdescribed in Great Britain Patent 2,220,211, which is incorporatedherein by reference.

U.S. Pat. No. 4,855,283 granted to Lockhoff et al. on Aug. 8, 1989,which is incorporated herein by reference, teaches glycolipid analoguesincluding N-glycosylamides, N-glycosylureas and N-glycosylcarbamates,each of which is substituted in the sugar residue by an amino acid, asimmuno-modulators or adjuvants. Lockhoff reported thatN-glycosphospholipids and glycoglycerolipids are capable of elicitingstrong immune responses in both herpes simplex virus vaccine andpseudorabies virus vaccine. Some glycolipids have been synthesized fromlong chain-alkylamines and fatty acids that are linked directly with thesugars through the anomeric carbon atom, to mimic the functions of thenaturally occurring lipid residues.

U.S. Pat. No. 4,258,029 granted to Moloney, incorporated herein byreference, teaches that octadecyl tyrosine hydrochloride (OTH) functionsas an adjuvant when complexed with tetanus toxoid and formalininactivated type I, II and III poliomyelitis virus vaccine. Lipidationof synthetic peptides has also been used to increase theirimmunogenicity.

Therefore, according to the invention, the immunogenic, antigenic,pharmaceutical, including vaccine, compositions comprising a PMPprotein, or a PMP derived polypeptide or a PMP encoding nucleic acid orfragment thereof, vector or cell expressing the same, may furthercomprise an adjuvant, such as, but not limited to alum, mLT, LTR192G,QS21, Ribi DETOX™, MMPL, CpG DNA, MF59, calcium phosphate, PLG and allthose listed above. Preferably, the adjuvant is selected from one ormore of the following: alum, QS21, CpG DNA, PLG, LT, 3D-mPL, or BacilleCalmette-Guerine (BCG) and mutated or modified forms of the above,particularly mLT and LTR192G. The compositions of the present inventionmay also further comprise a suitable pharmaceutical carrier, includingbut not limited to saline, bicarbonate, dextrose or other aqueoussolution. Other suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences, Mack Publishing Company, a standardreference text in this field, which is incorporated herein by referencein its entirety.

Immunogenic, antigenic and pharmaceutical, including vaccine,compositions may be administered in a suitable, nontoxic pharmaceuticalcarrier, may be comprised in microcapsules, microbeads, and/or may becomprised in a sustained release implant.

Immunogenic, antigenic and pharmaceutical, including vaccine,compositions may desirably be administered at several intervals in orderto sustain antibody levels and/or T-cell levels. Immunogenic, antigenicand pharmaceutical, including vaccine, compositions may be used inconjunction with other bacteriocidal or bacteriostatic methods.

Another embodiment of the vaccines of the present is a vaccinecomprising one or more isolated or purified PMPE or PMPI polypeptides orPMPE-derived polypeptides or PMPI-derived polypeptides, or homologsthereof, of Chlamydia spp, having a molecular weight between 90 and 115kDa as determined in SDS polyacrylamide gel electrophoresis, or isolatednucleic acids encoding a PMPE or PMPI polypeptide, or PMPE-derivedpolypeptide, or PMPI-polypeptide from Chlamydia spp., having a molecularweight between 90 and 115 kDa as determined in SDS polyacrylamide gelelectrophoresis, and further comprising one or more components selectedfrom the group consisting of alum, mLT, LTR192G, QS21, MF59, CpG DNA,MPL, calcium phosphate and PLG. Optionally, the vaccine may include HMWprotein or fragments thereof, C. trachomatis MOMP or fragments thereof,C. trachomatis PMPH or fragments thereof, or C. trachomatis HtrA orfragments thereof, or a combination of the foregoing. The compositionsmay optionally comprise trachomatis LPS or LPS from a recombinantbacteria which has been engineered to express lpxA or Kdo transferase ofC. trachomatis and which is defective in its own lpxA or Kdo transferasegene

Also included in the invention is a method of producing an immuneresponse in an animal comprising immunizing the animal with an effectiveamount of one or more of the PMP polypeptides or nucleic acid moleculesencoding the PMP polypeptides of the invention, compositions comprisingthe same and vaccines comprising the same. The PMP polypeptides, nucleicacids, compositions and vaccines comprising the PMP polypeptides of theinvention may be administered simultaneously or sequentially. Examplesof simultaneous administration include cases in which two or morepolypeptides, nucleic acids, compositions, or vaccines, which maybe thesame or different, are administered in the same or different formulationor are administered separately, e.g., in a different or the sameformulation but within a short time (such as minutes or hours) of eachother. Examples of sequential administration include cases in which twoor more polypeptides, nucleic acids, compositions or vaccines, which maybe the same or different, are not administered together or within ashort time of each other, but may be administered separately atintervals of, for example, days, weeks, months or years.

The polypeptides, nucleic acid molecules or recombinant bacterialvaccines of the present invention are also useful in the generation ofantibodies, as described supra, or T-cells. For T-cells, animals,including humans, are immunized as described above. Followingimmunization, PBL (peripheral blood lymphocytes), spleen cells or lymphnode cells are harvested and stimulated in vitro by placing largenumbers of lymphocytes in flasks with media containing human serum. Apolypeptide of the present invention is added to the flasks, and T-cellsare harvested and placed in new flasks with X-irradiated peripheralblood mononuclear cells. The polypeptide is added directly to theseflasks, and cells are grown in the presence of IL-2. As soon as thecells are shown to be Chlamydia specific T-cells, they are changed to astimulation cycle with higher IL-2 concentrations (20 units) to expandthem.

Alternatively, one or more T-cells that proliferate in the presence of apolypeptide of the present invention can be expanded in number bycloning. Methods for cloning cells are well known in the art. Forexample, T-cell lines may be established in vitro and cloned by limitingdilution. Responder T-cells are purified from the peripheral bloodestablished in culture by stimulating with the nominal antigen in thepresence of irradiated autologous filler cells. In order to generateCD4⁺ T-cell lines, the Chlamydia polypeptide is used as the antigenicstimulus and autologous PBL or lymphoblastoid cell lines (LCL)immortalized by infection with Epstein Barr virus are used as antigenpresenting cells. In order to generate CD8⁺ T-cell lines, autologousantigen-presenting cells transfected with an expression vector whichproduces the relevant Chlamydia polypeptide may be used as stimulatorcells. T-cell lines are established following antigen stimulation byplating stimulated T-cells in 96-well flat-bottom plates with PBL or LCLcells and recombinant interleukin-2 (rIL2) (50 U/ml). Wells withestablished clonal growth are identified at approximately 2-3 weeksafter initial plating and restimulated with appropriate antigen in thepresence of autologous antigen-presenting cells, then subsequentlyexpanded by the addition of low doses of IL2. T-cell clones aremaintained in 24-well plates by periodic restimulation with antigen andIL2 approximately every two weeks.

T-cell preparations may be further enriched by isolating T-cellsspecific for antigen reactivity using the methods disclosed by Kendrickset al. in U.S. Pat. No. 5,595,881.

The vaccine compositions of the present inventions are useful inpreventing, treating or ameliorating disease symptoms in an animal,preferably a human, with a disease or disorder associated with Chlamydiainfection or to prevent the occurrence or progression of a disease ordisorder associated with Chlamydia infection in an animal, preferably ahuman. Such diseases or disorders include, but are not limited to,Chlamydia bacterial infection, conjunctivitis, urethritis,lymphogranuloma venereum (LGV), cervicitis, epididymitis, salpingitis,tubal occlusion, infertility, cervical cancer, reactive arthritis,arteriosclerosis and atherosclerosis.

5.8. Immunoassays and Diagnostic Reagents

The PMP protein, PMP-derived polypeptide or nucleic acid encoding same,and fragments thereof, are useful as diagnostic reagents. Use of theproteins and nucleic acids of the invention as an antigen or immunogenfor the generation of anti-PMP protein antibodies or as an antigen inimmunoassays including enzyme-linked immunosorbent assays (ELISA),radioimmunoassays (RIA) and other non-enzyme linked antibody bindingassays or procedures known in the art for the detection ofanti-bacterial, anti-Chlamydia, and anti-PMP protein antibodies areencompassed by the invention.

ELISA is well-known and routine in the art. Generally, in ELISA, the PMPprotein is immobilized onto a selected surface, for example, a surfacecapable of binding proteins such as the wells of a polystyrenemicrotiter plate. After washing to remove incompletely absorbed PMPprotein, a nonspecific protein solution that is known to beantigenically neutral with regard to the test sample may be bound to theselected surface. This allows for blocking of nonspecific absorptionsites on the immobilizing surface and thus reduces the background causedby nonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials, to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of bovine gamma globulin(BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is thenallowed to incubate for from 2 to 4 hours, at temperatures on the orderof about 20° C. to 37° C. Following incubation, the sample-contactedsurface is washed to remove non-immunocomplexed material. The washingprocedure may include washing with a solution, such as PBS/Tween or aborate buffer. Following formation of specific immunocomplexes betweenthe test sample and the bound PMP protein, and subsequent washing, theoccurrence, and even amount, of immunocomplex formation may bedetermined by subjecting the immunocomplex to a second antibody havingspecificity for the first antibody. If the test sample is of humanorigin, the second antibody is an antibody having specificity for humanimmunoglobulins and in general IgG.

To provide detecting means, the second antibody may have an associatedactivity such as an enzymatic activity that will generate, for example,a color development upon incubating with an appropriate chromogenicsubstrate. Detection may then be achieved by detecting color generation.Quantification may then be achieved by measuring the degree of colorgeneration using, for example, a visible spectrophotometer and comparingto an appropriate standard. Any other detecting means known to thoseskilled in the art are included.

In Western blot assays, the polypeptide, either as a purifiedpreparation or a cell extract, is subjected to SDS-PAGE electrophoresis,for example, as described by Laemmli, Nature 227:690 (1970) or any othermethod known in the art. After transfer to a nitrocellulose membrane,the material is further incubated with the serum sample, polyclonalantibody preparation, or monoclonal antibody diluted in the range offrom about 1:5 to 1:5000, preferably from about 1:100 to about 1:500,depending upon the titer and specification of the antibodies.Appropriate dilutions can be readily determined by methods known in theart. The reaction is revealed according to standard procedures. Forexample, when human antibody is used, the membrane is incubated in agoat anti-human peroxidase conjugate for an appropriate length of time.The membrane is washed. The reaction is developed with the appropriatesubstrate and stopped. The reaction is measured visually by theappearance of a colored band e.g., by colorimetry.

In a dot blot assay, the purified or partially purified polypeptide orcell extract can be used. Briefly, a solution of the antigen at about100 μg/ml is serially two-fold diluted in 50 mM Tris-HCl (pH 7.5). 100μl of each dilution is applied to a 0.45 μm nitrocellulose membrane setin a 96-well dot blot apparatus. The buffer is removed by applyingvacuum to the system. Wells are washed by addition of 50 μM Tris-HCl (pH7.5) and the membrane is air-dried. The membrane is saturated in blockbuffer (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl and 10 g/L skim milk) andincubated with an antiserum dilution from about 1:50 to about 1:500. Thereaction is revealed according to standard procedures. For example, agoat anti-rabbit peroxidase conjugate is added to the well when rabbitantibodies are used. Incubation is carried out 90 minutes at 37° C. andthe blot is washed. The reaction is developed with the appropriatesubstrate and stopped. The reaction is measured visually by theappearance of a colored spot, e.g., by colorimetry.

The PMP proteins, PMP-derived polypeptides or nucleic acids encodingsame, and fragments thereof, are also useful as antigens or immunogensfor the generation of anti-PMP protein T-cell response or as an antigenin immunoassays, including T-cell proliferation assays, cytokineproduction, delayed hypersensitivity reactions or cytotoxic T-cells(CTL) reactions.

For analysis of Chlamydia peptide specific T-cell proliferativeresponses, fresh peripheral blood, spleen or lymph node cells areharvested. Cells are plated into 96-well round bottom microtiter platesand are incubated with peptides. Data is expressed as a stimulationindex (SI) which is defined as the mean of the number of cells inexperimental wells divided by the mean of the number of cells in controlwells (no antigen).

For analysis of cytokine release of T-cells in response to Chlamydiapolypeptides, responder cells are mixed with polypeptides. Supernatantsare collected and added to an ELISA coated with antibody to the cytokine(e.g., anti-IFN-α or anti-IL-2 antibody). After washing, rabbitanti-cytokine polyclonal antibody (e.g., anti-IFN-α or anti-IL-2) isadded. Labeled goat anti-rabbit IgG polyclonal is added. Substrate isadded and the amount of cytokine released into the supernatant isdetermined based upon the amount of color developed in the ELISA.

Another embodiment includes diagnostic kits comprising all of theessential reagents required to perform a desired immunoassay accordingto the present invention. The diagnostic kit may be presented in acommercially packaged form as a combination of one or more containersholding the necessary reagents. Such a kit may comprise PMP protein,PMP-derived polypeptide or nucleic acid encoding same, or a monoclonalor polyclonal antibody of the present invention, in combination withseveral conventional kit components. Conventional kit components will bereadily apparent to those skilled in the art and are disclosed innumerous publications, including, for example, Harlow and Lane,Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. 1988) which is incorporated herein by referencein its entirety. Conventional kit components may include such items as,for example, microtiter plates, buffers to maintain the pH of the assaymixture (such as, but not limited to Tris, HEPES, etc.), conjugatedsecond antibodies, such as peroxidase conjugated anti-mouse IgG (or anyanti-IgG to the animal from which the first antibody was derived) andthe like, and other standard reagents.

The nucleic acid molecules containing the PMP encoding sequences of thepresent invention may be used in combination with an appropriateindicator means, such as a label, for determining hybridization. A widevariety of appropriate indicator means are known in the art, includingradioactive, enzymatic or other ligands, such as avidin/biotin anddigoxigenin-labeling, which are capable of providing a detectablesignal. In some diagnostic embodiments, an enzyme tag, such as urease,alkaline phosphatase or peroxidase, instead of a radioactive tag, may beused. In the case of enzyme tags, colorimetric indicator substrates areknown which can be employed to provide a means visible to the human eyeor spectrophotometrically, to identify specific hybridization withsamples containing PMP protein gene sequences.

Probes of the invention can be used in diagnostic tests, as capture ordetection probes. Such capture probes can be conventionally immobilizedon a solid support directly or indirectly, by covalent means or bypassive adsorption. A detection probe can be labeled by a detectionmarker selected from radioactive isotopes, enzymes, such as peroxidase,alkaline phosphatase, and enzymes able to hydrolyze a chromogenic,fluorogenic or luminescent substrate; compounds that are chromogenic,fluorogenic or luminescent; nucleotide base analogs; and biotin.

Probes of the invention can be used in any conventional hybridizationtechniques, such as dot blot (Maniatis et al., Molecular Cloning: ALaboratory Manual (1982) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. 1982), Southern blot (Southern, J. Mol. Biol. 98:5031975), northern blot (identical to Southern blot to the exception thatRNA is used as a target), or sandwich techniques (Dunn et al., Cell12:23 1977).

In embodiments involving solid-phase procedures, the test DNA (or RNA)from samples, such as clinical samples, including exudates, body fluids(e.g., serum, amniotic fluid, middle ear effusion, sputum, semen, urine,tears, mucus, bronchoalveolar lavage fluid) or even tissues, is absorbedor otherwise affixed to a selected matrix or surface. The fixed,single-stranded nucleic acid is then subjected to specific hybridizationwith selected probes comprising the nucleotide sequences encoding a PMPprotein, or fragments or analogues thereof, under desired conditions.The selected conditions will depend on the particular criteria requiredand on, for example, the G+C content, type of target nucleic acid,source of nucleic acid, size of hybridization probe, etc. Followingwashing of the hybridization surface so as to remove non-specificallybound probe molecules, specific hybridization is detected, or evenquantified, by means of the label. It is preferred to select nucleotideacid sequence portions that are conserved among species of Chlamydia.The selected probe may be at least 15 bp and may be in the range ofabout 30 to 90 bp.

The invention also relates to methods for identifying compounds whichinteract with and inhibit or activate an activity of the polypeptides ornucleic acid molecules of the invention comprising contacting acomposition comprising the polypeptide or the nucleic acid molecule withthe compound to be screened under conditions that permit interactionbetween the compound and the polypeptide or nucleic acid molecule toassess the interaction of a compound and to detect interaction of thecompound with the polypeptide of nucleic acid. The interaction of thecompound with the polypeptide or nucleic acid molecule is determined bythe association of a second component (e.g., an antibody) capable ofproviding a detectable signal in response to the interaction of thepolypeptide or nucleic acid molecule with the compound; and determiningthe presence or absence of a signal generated from the interaction ofthe compound with the polypeptide or nucleic acid molecule.Alternatively, the interaction of the compound with the polypeptide ornucleic acid molecule is determined by the ability of the compound toinhibit the activity of the polypeptide or the nucleic acid molecule.Thus, the invention also provides agonists and antagonists of the PMPpolypeptides of the invention.

5.9. Applications

The proteins, polypeptides, peptides, antibodies, T-cells and nucleicacids of the invention are useful as reagents for clinical or medicaldiagnosis of Chlamydia infections and for scientific research on theproperties of pathogenicity, virulence, and infectivity of Chlamydia, aswell as host defense mechanisms. For example, DNA and RNA of theinvention can be used as probes to identify the presence of Chlamydia inbiological specimens by hybridization or PCR amplification. The DNA andRNA can also be used to identify other bacteria that might encode apolypeptide related to the Chlamydia PMP protein. The proteins of theinvention may be used to prepare polyclonal and monoclonal antibodiesthat can be used to further purify compositions containing the proteinsof the invention by affinity chromatography or for use as diagnostic oras prophylactic or therapeutic agents. The proteins can also be used instandard immunoassays to screen for the presence of antibodies orT-cells to Chlamydia in a biological sample.

5.10. Biological Deposits

Certain plasmids that contain portions of the gene having the openreading frame of the PMP genes encoding the PMP proteins of the presentinvention have been inserted into E. coli and deposited with theAmerican Type Culture Collection (ATCC) located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209, U.S.A., pursuant to the BudapestTreaty and pursuant to 37 CFR 1.808 and prior to the filing of thisapplication. All restrictions imposed on the availability of thedeposited material will be irrevocably removed upon grant of a patentbased upon this United States patent application. The inventiondescribed and claimed herein is not to be limited by the scope of theplasmids deposited, since the deposited embodiment is intended only asan illustration of the invention. Any equivalent or similar plasmidsthat encode similar or equivalent proteins or fragments or analoguesthereof as described in this application are within the scope of theinvention. Plasmid ATCC Accession No. Date Deposited M15 pREP(pQE-pmpE)#37 ATCC PTA-2462 Sep. 12, 2000 TOP10(pBAD-pmpI-Ct-Uni)#7 ATCCPTA-2461 Sep. 12, 2000

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for purposes ofillustration and not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein such terms are intended in a descriptive senseand not for purposes of limitation.

6. EXAMPLES

The above disclosure generally describes the present invention. Theexamples are described solely for the purpose of illustration and arenot intended to limit the scope of the invention. Changes in form andsubstitution of equivalents are contemplated as circumstances suggest orrender expedient. Although specific terms have been employed herein,such terms are intended in a descriptive sense and not for purposes oflimitation.

Methods of molecular genetics, protein biochemistry and immunology usedbut not explicitly described in the disclosure and examples are amplyreported in the scientific literature and are well within the ability ofthose skilled in the art.

6.1. Extraction of Envelope Proteins

McCoy cells are cultured either in standard 225 cm² tissue cultureflasks or in Bellco spinner flasks (Cytodex microcarrier, Pharmacia) at37° C. in 5% CO₂ using DMEM media supplemented with 10%Chlamydia-antibody-free fetal bovine serum, glucose and nonessentialamino acids. C. trachomatis, including but not limited to the L2 serovar(ATCC VR-902B), elementary bodies (EBs) are prepared from lysates ofinfected McCoy cells. Basically, McCoy cells infected with C.trachomatis are sonicated and cellular debris is removed bycentrifugation. The supernatant containing Chlamydial EBs is thencentrifuged and the pellet containing the EBs is resuspended in Hanks'balanced salts solution (HBSS). RNAase/DNAase solution is added andincubated at 37° C. for 1 hour with occasional mixing. The EB containingsolution is layered onto a discontinuous density gradient (40%, 44% and54%) of Renogratin-60 (mixture of diatrizoate melgumine and diatrizoatesodium, Bracco Diagnostics, Princeton, N.J.) and ultracentrifuged forseparation of the EBs on the gradient. After centrifugation, the EBs areharvested from the gradient between the interface of the 44% and 54%layers. The EBs are washed in phosphate buffered saline and resuspendedin HBSS.

Purified EBs are sequentially extracted with 0.1% OGP [high ionicstrength] in HBSS to remove peripheral surface proteins and held on ice.The same EB preparation is then extracted with 1.0% OGP, 10 mM DTT, 1 mMPMSF, and 10 mM EDTA, in a 50 mM Tris pH 7.4 buffer. The extracts aredialyzed (3500 molecular weight cut off) to remove detergent and otherreagents and are concentrated by lyophilization. Eluents are dialyzed toremove excess detergent and then lyophilized. Envelope proteins are sizefractionated by SDS-PAGE and visualized by silver staining or analyzedby Western blotting. Protein(s) of about 90-115 kDa present in moderateamounts are detected in the gel.

6.2. Amino Terminal Sequencing of PMP Polypeptide

To obtain the N-terminal amino acid sequence, sufficient quantities ofthe PMPE or PMPI protein (≧5 μg) are electroblotted onto a PVDF membrane(Applied Biosystems), and stained with Coomassie blue. Immobilizedprotein is released from the membrane and treated in situ with lowlevels of endopeptidase Lys-C, endopeptidase Arg-C and/or endopeptidaseGlu-C to fragment the native protein. The resulting peptide fragmentsare purified by HPLC and their N-terminal amino acid sequences aredetermined using an ABI 430 Protein Sequenator and standard proteinsequencing methodologies.

6.3. Determination of Post-Translational Modifications

Recently, several C. trachomatis membrane-associated proteins have beenshown to be post-translationally modified. The 18 kDa and 32 kDacysteine-rich EB proteins, which are lectin-binding proteins, have beenshown to carry specific carbohydrate moieties (Swanson et al. 1990.Infect. Immun. 58:502-507). Incorporation of radiolabeled palmitic acidhas been used to demonstrate that the about 27 kDa C. trachomatisMip-like protein is lipidated (Lundemose et al. 1993. J. Bacteriol.175:3669-3671). Swanson et al. have discovered that the MOMP from the L2serovar contains N-acetylglucosamine and/or N-acetylgalactosamine andthese carbohydrate moieties mediate binding of MOMP to Hela cellmembranes.

To ascertain whether the PMPE or PMPI protein is glycosylated, EBs aregrown on McCoy cells in the presence of tritiated galactose orglucosamine and analyzed by SDS-PAGE and autoradiography. Briefly, McCoycells are grown in T225 flasks under standard conditions (DMEM+10% FCS,35 ml per flask, 10% CO₂) to about 90% confluency and inoculated withsufficient EBs to achieve 90%-100% infectivity. Following a 3 hourinfection period at 37° C. cycloheximide is added (1 μg/ml) to inhibithost cell protein synthesis and the cultures reincubated for anadditional 4-6 hours. Approximately 0.5 mCi of tritiated galactose(D-[4,5-³H(N)]galactose, NEN) or glucosamine (D-[1,6-³H(N)glucosamine,NEN) is then be added to each flask and the cultures allowed to incubatefor an additional 30-40 hours. Cells are harvested by scraping, and EBspurified by gradient centrifugation. PMPE or PMPI protein is isolatedfrom 1.0% OGP surface extracts, eluted with NaCl and analyzed bySDS-PAGE using ¹⁴C-labeled molecular weight markers (BRL). The resultinggel is dried and subjected to autoradiography by exposure for 1-4 weeksto Kodak X-AR film at −70° C.

To determine post synthesis lipid modification, C. trachomatis iscultivated on monolayers of McCoy cells according to standardprocedures. Approximately 24 hours postinfection, conventional culturemedia (DMEM+10% FCS) is removed and replaced with a serum-free mediumcontaining cycloheximide (1 μg/ml) and [U-¹⁴C]palmitic acid (0.5mCi/T225 flask, NEN) and incubated for a further 16-24 hours to allowprotein lipidation to occur. Surface EB extracts are prepared andanalyzed by autoradiography as described above.

6.4. Anti-PMPE or Anti-PMPI Antiserum

Antisera to PMPE or PMPI polypeptides are prepared by injecting the PMPEor PMPI polypeptide into an animal, such as a rabbit, mouse or guineapig, with or without an adjuvant by any method generally known to thoseskilled in the art. For instance, PMPE is injected with Freund'scomplete adjuvant followed by injections of PMPE with Freund'sincomplete adjuvant. Normally, a semi-purified or purified form of theprotein is injected. For instance, the PMPE polypeptide is resolved fromother proteins using SDS-PAGE according to standard techniques wellknown to those skilled in the art, as previously described (Laemmli,1970, Nature 227:680-685), and cutting the PMPE-containing band out ofthe gel. The excised band containing PMPE is macerated and injected intoan animal to generate antiserum to the PMPE polypeptide. The antisera isexamined using well known and generally accepted methods of ELISA todetermine titer, Western blots to determine binding to proteins, and forimmunofluorescent staining and for complement-mediated cytotoxicactivity against Chlamydia.

To aid in the characterization of the PMPE or PMPI protein, hyperimmunerabbit antisera is raised against whole EBs from C. trachomatis. Eachanimal is given a total of three immunizations of about 250 μg ChlamydiaEBs per injection (beginning with the EBs mixed with complete Freund'sadjuvant and followed with EBs mixed with incomplete Freund's adjuvant)at approximately 21 day intervals. At each immunization, approximatelyhalf of the material is administered intramuscularly (i.m.) and half isinjected intranodally. Fourteen days after the third vaccination, afourth booster of about 100 μg of EBs is given i.m. and the animalsexsanguinated 7-10 days later.

6.5. ELISA

Anti-PMPE or anti-PMPI antibody titers are measured by ELISA usingpurified PMPE or PMPI protein (˜1 μg/well) or C. trachomatis EBs (wholeor crude protein extracts) or cells infected with Chlamydia as captureligands by any method known by those skilled in the art. Serialdilutions of antisera are made in PBS and tested by ELISA in duplicate.HRP-conjugated antibody is diluted and used as the second reporterantibody in these assays. Titers are expressed as the greatest dilutionshowing positive ELISA reaction, i.e., an O.D. 450 value>2SD above themean negative control value (pre-bleed rabbit sera).

6.6. Western Blots

Chlamydia trachomatis is grown in McCoy cells and Chlamydia cell lysatesare prepared as described in section 6.1, supra. The solubilized cellsare resolved on 12% polyacrylamide gels and the separated proteins wereelectrophoretically transferred to PVDF membranes at 100 V for 1.5 hoursas previously described (Thebaine et al. 1979, Proc. Natl. Acad. Sci.USA 76:4350-4354). The PVDF membranes are then pretreated with 25 ml ofDulbecco's phosphate buffered saline containing 0.5% sodium casein, 0.5%bovine serum albumin and 1% goat serum. All subsequent incubations arecarried out using this pretreatment buffer.

PVDF membranes are incubated with 25 ml of a dilution of preimmunerabbit serum or serum from a rabbit immunized with PMPE or PMPIpolypeptide (as described above) for 1 hour at room temperature or withmonoclonal antibodies to PMPE or PMPI. PVDF membranes are then washedtwice with wash buffer (20 mM Tris buffer [pH 7.5.] containing 150 mMsodium chloride and 0.05% Tween-20). PVDF membranes are incubated with25 ml peroxidase-labeled goat anti-rabbit (or anti-mouse formonoclonals) IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.)for 30 minutes at room temperature. PVDF membranes are then washed 4times with wash buffer, and are developed with 3,3′diaminobenzidinetetra-hydrochloride and urea peroxide as supplied by Sigma Chemical Co.(St. Louis, Mo.; catalog number D-4418) for 4 minutes each.

Hyperimmune antisera or monoclonal antibody is used to probe Westernblots of crude EB or RB extracts as well as 1.0% OGP EB extractpreparations to identify PMPE or PMPI polypeptides from other C.trachomatis serovars and Chlamydia species. Lysates from C. trachomatisA, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, L3, or MoPnserovars or Chlamydia pneumoniae are electrophoresed to identifyproteins reactive with antisera generated against PMPE or PMPI protein.

6.7. Cellular Envelope Location of PMPE and PMPI

Surface localization of the PMPE or PMPI protein on different Chlamydiastrains and derivatives is examined by indirect fluorescence antibody(IFA). IFA is performed using the procedures generally known by thoseskilled in the art using hyperimmune anti-PMPE or PMPI protein as theprimary antibody. Hak cells infected with whole EBs from C. trachomatisare achieved by the following method.

McCoy or Hak cells are grown to confluence in D-MEM media on 12 mm plaincoverslips inside 24 well tissue culture plates then centrifugallyinoculated with ˜5×10⁴ inclusion forming units (IFU) of the C.trachomatis. After ˜24 hours incubation, the culture media is removedand infected cells fixed in methanol for 10 min. The fixed monolayer isthen washed with PBS (1×) to remove fixative and overlayered with >300 lof anti-PMPE or PMPI rabbit antibody that has been diluted in PBS. After1 hour incubation with the primary antibody, the cells are washed gentlywith PBS then incubated for ˜30 minutes with mouse anti-rabbit IgGantibody conjugated with FITC. The second antibody is diluted using aPBS solution containing 0.0091% Evans Blue as a counter stain tovisualize the monolayer. Cells are washed 2× in PBS to remove thesecondary antibody, the coverslips are removed from the culture plates,and mounted onto microscope slides using a fluorescent mounting medium.

Identical cell samples are stained with pre-bleed rabbit antibody orFITC-conjugated second antibody alone are processed in parallel andserved as antibody specificity (negative) controls. Counterstainedsamples are examined at a 1000-× magnification with a Zeiss Axioskopphotomicroscope equipped with plan-neoflur objectives.

6.8. Properties of PMPE and PMPI Polypeptides

PMPE polypeptide exists as a protein of approximately 90-115 KDa in itsnative state as determined via Western blots of crude EB or RB extractsof Chlamydia, as described in Section 6.1. PMPI polypeptide exists as aprotein of approximately 90-115 KDa in its native state as determinedvia Western blots of crude EB or RB extracts of Chlamydia.

The isoelectric point of the PMPE protein is about 7.17. The isoelectricpoint of the PMPI protein is about 6.36.

6.9. Vaccine Efficacy

An in vitro neutralization model using methods generally known to thoseskilled in the art is used to show that protective antiserum inhibitsChlamydial infection (neutralization) of various tissue culture celllines. Animal models are also essential for testing vaccine efficacywith both small animal (non-primate) and primate models necessary forpreclinical evaluation. The guinea-pig is used for studying experimentalocular and genital infection by the Guinea-pig inclusion conjunctivitisagent (GPIC), C. psittaci.

The mouse offers a consistent and reproducible model of genital tractinfection using human genital tract isolates. This mouse model is agenerally accepted pre-clinical assay and was used to evaluate MOMP as asubunit vaccine. Another model is known as the primate model of trachomainfection wherein the induction of secretory IgA was shown to be a primecomponent of protection. Vaccinogenic ability of new subunit antigencandidates is determined using the above-mentioned generally accepted invitro neutralization and animal model systems.

6.9.1 In Vitro Neutralization Model

As a preliminary exercise to the animal protection studies, hyperimmuneanti-PMPE or PMPI antibody is evaluated for its ability to block theinfectivity of various C. trachomatis serovars (e.g., L2, B, F) invitro. Although McCoy cells are used to propagate Chlamydia, these cellsalso allow antibody-mediated uptake via Fc receptors. Therefore, toevaluate anti-PMPE or anti-PMPI antibody inhibition of infectivity, Hakcells, which do not display Fe receptors, are used in these analyses.

Cells are grown on coverslips in 24-well plates to a subconfluentmonolayer (about 90% confluency=1×10⁵ cells/ml) at 37° C. in 5% CO₂.Anti-PMPE or PMPI antibody is diluted to about 100 μg/ml (total protein)in sucrose-phosphate-glutamate (SPG) buffer and then serially diluted inSPG buffer. Frozen aliquots of pretitered Chlamydia EBs are diluted inSPG buffer to about 2×10⁴ IFU (inclusion forming units)/ml. EBs arepremixed with the diluted anti-PMPE or PMPI antibody and incubated 30minutes at 37° C. on a rocking platform.

Prepared Hak cells are washed in HBSS and then incubated with theanti-PMPE or PMPI antibody/Chlamydia EB mixture in triplicate for eachantibody using ˜500 IFU/ml. Plates are centrifuged at ˜500 rpm at roomtemperature for 1 hour then incubated for 2 hours at 37° C. or areincubated for 2 hours at 37° C. without prior centrifugation to allow EBinfection of the monolayer. Then the inoculum is removed and plateswashed 3 times with HBSS. Tissue culture media containing 1 μg/ml ofcycloheximide is added and plates incubated for about 24-48 hours at 37°C. in 5% CO₂ to allow inclusion bodies to develop. After incubation, themedia is removed and cell monolayers washed 3× in PBS. Plates are fixedin methanol for 20 minutes and re-washed in PBS.

Cells are stained to visualize inclusions by incubating withanti-Chlamydia LPS antibody (diluted about 1:500), washed 3 times inPBS, followed by incubation with FITC-conjugated goat secondary antibodyfor 30 minutes at 37° C. Coverslips are washed, air dried, and mountedin glycerol on glass slides. Inclusions are counted in five fieldsthrough the midline of the coverslip on a Zeiss fluorescencephotomicroscope. Results are reported as the percent reduction ofinclusion-containing cells with respect to a heterogenous antibodycontrol (rabbit pre-bleed sera).

6.9.2 Vaccine Efficacy (Mouse Model of Salpingitis and Fertility)

Immunization and Challenge

The Tuffrey murine infertility model is employed to evaluate theefficacy of rPMPE or rPMPI to protect animals against Chlamydiatrachomatis-induced salpingitis and infertility. Three groups of femaleC3HeOuJ mice (6 weeks of age, Jackson Labs) are employed for thisevaluation. The test group is immunized (e.g., intradermally,intraperitoneal, subcutaneously, intramuscularly, or mucosally) byadministration of a vaccine formulation containing approximately 10-100μg of purified rPMPE or rPMPI and adjuvant. For instance, mice that areimmunized intranasally are sedated using an anesthesia cocktailconsisting of 16% Ketaject and 16% Xlaject in 68% pyrogen-free PBS (100μl) i.p./animal. Sedated animals are placed on their backs and using astandard laboratory pipette administered the vaccine formulations;approximately 10 μl of the vaccine solution per nostril with a 5-10minute wait period between applications.

Two groups of female mice are immunized similarly but with a preparationcontaining adjuvant only, no antigen. One of these groups issubsequently challenged with C. trachomatis (sham immunized, infected)and serves as the negative fertility control while the other group isnot challenged (sham immunized, sham infected) and serves as thepositive fertility control.

At week 4, all animals are administered a single i.p. dose ofprogesterone (2.5 mg in pyrogen-free PBS, Depo-Provera, Upjohn) tostabilize the uterine epithelium. At week 5, animals immunized withrPMPE or rPMPI and animals in the negative control group are infected bybilateral intrauterine inoculation with approximately 5×10⁵ IFU of C.trachomatis (including but not limited to, from serovar F, strain NI1)in 100 μl of sucrose phosphate glutamate buffer (SPG). To mimic themanipulations to the reproductive tract experienced by the other groups,animals in the positive control are bilaterally inoculated with 100 μlof a McCoy cell extract that contains no C. trachomatis. At week 7,animals from each group are sacrificed by CO₂ asphyxiation and thecomplete genital tract (both upper and lower reproductive tracts) areremoved for histopathological analysis. At week 9, the remaining femalesfrom each group are caged with 8-10 week old male C3H mice for a 2 monthbreeding period to assess fertility (1 male for every 2 females per cagewith weekly rotation of the males within each group, animals fromdifferent experimental groups were not mixed). Palpation and periodicweighing are used to determine when animals in each pair becomepregnant. The parameters used to estimate group fertility are: F, thenumber of mice which littered at least once during the mating perioddivided by the total number of mice in that study group; M, the numberof newborn mice (born dead or alive) divided by the number of littersproduced in that group during the mating period; and N, the number ofnewborn mice (born dead or alive) divided by the total number of mice inthat group.

Histopathology

Genital tracts are treated for >24 hours in Bouin's fixative,progressively dehydrated in 50%, 70% and 100% methanol, soaked intoluol, and either paraffin embedded or directly embedded in OCTcompound (Tissue-TEK, Miles) and subsequently snap frozen in liquidnitrogen. Tissue sections (approximately 6 μm) are stained withhematoxylin and eosin (after deparaffinization of the Bouin fixedsamples). Inflammatory changes in the oviducts and ovaries are graded asfollows: 0, no apparent inflammatory reaction; 1, a few mononuclearcells infiltrating the periovarial space or the submucosa of theoviduct; 2, same as 1 but to a greater extent; 3, same as 2 but with athickened oviductal submucosa and the presence of inflammatory cells inthe oviductal lumen; 4, same as 3 but to a greater extent. Inflammationin the cervix/vagina is scored based on the level of the intraepithelialinfiltrate observed.

Determination of rPMPE or rPMPI Specific Humoral Responses

Blood samples are collected periodically during the immunization andchallenge phases by retroorbital bleeding and serum prepared bycentrifugation. Vaginal secretions are collected by repeated injectionof 50-100 μl of sterile PBS into the vagina with a standard laboratorypipetter and immediately withdrawing the solution. Two to threeinjection/withdrawal cycles are performed.

Quantitation of antibody (Ab) responses by ELISA are performed asdescribed in Section 6.5. Microwell ELISA plates (Maxisorb, NUNC) fordetermining antibody levels are coated overnight at 4° C. with ˜0.5-1.0μg of purified rPMPE or rPMPI per well in 10 mM carbonate/bicarbonatebuffer (pH 9.6), washed with PBS containing 0.1% Tween-20 (washingbuffer) and blocked for ˜1 hr at 37° C. with a PBS solution containing3% BSA. For the determination of antigen-specific serum IgG levels, testsera or vaginal secretions are serially diluted in washing buffercontaining 0.5% BSA and aliquots (100 (l) incubated in theantigen-coated wells for ˜2 hr at 37° C. The plates are then washed andincubated for ˜1 hr at 37° C. with a horseradish peroxidase(HRP)-conjugated goat anti-mouse IgG second antibody (Sigma). AHRP-conjugated goat anti-mouse IgA secondary antibody is used to detectthe presence of rPMPE or rPMPI specific IgA in serum or vaginalsecretions. After incubation with the appropriate secondary Ab, theplates are washed and incubated for ˜20-30 minutes at room temperaturewith TMB substrate (Sigma). Reactions are stopped by the addition of 2MH₂SO₄ and the absorbance determined at 450 nm on a Molecular DevicesSpectroMax microplate reader. Titers are determined as the reciprocal ofthe sample dilution corresponding to an optical density of 1.0 at 450nm.

Determination of rPMPE or rPMPI Specific Cellular Responses

Groups of mice are immunized with rPMPE or rPMPI plus adjuvant vaccineas described above. At week 5, animals from each group are sacrificed byCO₂ asphyxiation, spleens removed and single cell suspensions preparedusing conventional methodologies. For cellular assessment, animals arenot treated with hormone. For both the positive control group (shamimmunized and sham infected) and the negative control group (shamimmunized, infected), spleen cells are pooled and tested forrestimulation.

For the measurement of spleen cell proliferation, spleens are ground (5to 10 rounds) in 5 ml RPMI 1640 Glutamax I supplemented with 10% fetalcalf serum, 25 mM HEPES, 50 U/ml penicillin, 50 μg/ml streptomycin, 1 mMsodium pyruvate, nonessential amino acids, and 50 mM 2-mercaptoethanol(Gibco-BRL). Live cells are counted by Trypan Blue staining and dilutedin the same media to reach a density of 1.0-2.0×10⁶ cells/ml (Falcon2063 polypropylene tubes). Triplicate cultures are set-up in roundbottom 96-well culture plates (Nunclon, Nunc) using ˜5×10⁵ respondercells per well in 200 μl of media. Cells are stimulated with rPMPE orrPMPI (antigen-specific proliferation) or with concanavalin A(Boerhinger Mannheim) as a positive stimulation control; unstimulatedcell cultures are used as a negative control of cellular activation.After 72-96 hours of incubation at 37° C. in 5% CO₂, cells are pulsedlabeled for ˜18 hrs with 1.0 Ci ³H-thymidine (Amersham) per well. Pulsedcells are harvested onto glass-fiber sheets using a Tomtec CellHarvester (Mk III) and counted for beta-emission in a 3-channel Wallac1450 Trilux Liquid Scintillation Counter. The stimulation index (SI) fora sample (individual or pooled) is defined as the mean of the antigen orConA-stimulated T-cell uptake of ³H-thymidine for triplicate wellsdivided by the mean of the unstimulated uptake for triplicate wells. SIsfor both antigen-specific (rPMPE or rPMPI-specific) and ConA-specificproliferation are determined.

Results presented in FIG. 7 demonstrate that animals immunized withrecombinant PMPE protein develop a strong and uniform antigen-specificT-cell proliferative response. These data also show that T-cells fromanimals immunized with recombinant PMPE recognize and are stronglystimulated by the infectious EB form of C. trachomatis.

6.10. Isolation of C. Trachomatis Chromosomal DNA

Chlamydia trachomatis were grown as described supra in Section 6.1.Gradient purified EBs were suspended in ˜5.0 ml of sterile water. Anequal volume of lysis buffer (200 mM NaCl, 20 mM EDTA, 40 mM Tris-HCl pH8.0, 0.5% (w/v) SDS, 0.5% (v/v) 2-mercaptoethanol, and 250 μg/ml ofproteinase K) was added and the cells suspended by gentle agitation andtrituration. The cell suspension was then incubated ˜12 hours at 50° C.to lyse the EBs and liberate chromosomal DNA. Proteinaceous material wasprecipitated by the addition of 5.0 ml of saturated NaCl (˜6.0 M, insterile water) and centrifugation at ˜5,500×g in a Sorval SS34 rotor atroom temperature. Chromosomal DNA was precipitated from the clearedsupernatant by the addition of two volumes of 100% ethanol. AggregatedDNA was collected and washed using gentle agitation in a small volume ofa 70% ethanol solution. Purified chromosomal DNA was suspended insterile water and allowed to dissolve/disburse overnight at 4° C. bygentle rocking. The concentration of dissolved DNA was determinedspectrophotometrically at 260 nm using an extinction coefficient of 1.0O.D. unit ˜50 μg/ml.

6.11. PCR Cloning of the PMPE ORF

To produce high levels of recombinant PMPE protein for immunogenicityand protective efficacy studies, the PMPE ORF was PCR cloned into an E.coli high expression vector, pQE-30 (QiaGen). Genes cloned into thepQE-30 vector are expressed from a T5 promoter which is under thecontrol of the lac operator. Genes cloned into pQE-30 are expressed as afusion protein containing a Met-Arg-Gly-Ser-(His)₆ (SEQ ID NO.:71) Nterminus. An additional 15 amino acid segment encoded by vectorsequences residing between the (His)₆ domain and the SalI site are alsopart of the N terminus. Oligonucleotide PCR primers complementary to theDNA sequences encoding the first 10 amino acid residues of theN-terminus of the mature protein and the last 9 C-terminal amino acidresidues of the C. trachomatis serovar L2 PMPE ORF present in the UCBerkeley genomic database (see Stephens et al., 1998, Science: 282:754-759 and GenBank Accession No. NC000117) were synthesized. Inaddition to the PMPE specific sequences, these PCR primers were designedto contain flanking SalI restriction sites to allow cloning of the ORFinto the pQE-30 expression vector. The oligonucleotides (SEQ ID NOs.:66and 67) were used to amplify PMPE-specific PCR products from C.trachomatis L2 serovar, genomic DNA.

The sequence of the amplification primers for SEQ ID NO.:66 (forwardprimer) and SEQ ID NO.:67 (reverse primer) used for these PCR reactionsare shown below. Forward primer for PMPE (SEQ ID NO.: 66.) 5′- ATC CAGCAG AG GGT CGA CGG GTT CCA GAT CCT ACG AAA GAG TCG CTA TC- 3′ Reverseprimer for PMPE (SEQ ID NO.: 67) 5′- ATC CAG CAG AGG GTC GAC GGC C TTAGAA TCG CAG AGC AAT TTC CCC ATT GA- 3′

In addition to the PMPE coding sequences, a TAA (TAA in reversecomplement) stop codon was engineered into the reverse primerimmediately after the last PMPE codon to end protein synthesis. A SalIrestriction site was engineered into the reverse primer downstream andadjacent to the TAA stop codon to facilitate cloning into pQE-30.

Standard PCR amplification reactions (2 mM Mg²⁺, 200 μmol dNTPs, 0.75units AmpliTaq, 50 l final volume) were programmed using ˜0.1 μg of C.trachomatis L2 chromosomal DNA. Amplification of the PMPE targetsequence was achieved using a standard 32-cycle, three-step thermalprofile, i.e., 95° C., 30 sec; 55° C., 45 sec; 72° C., 3 min.Amplification was carried out in 0.2 ml polypropylene thin-walled PCRtubes (Perkin-Elmer) in a Perkin-Elmer model 2400 thermal cycler. PCRamplification reactions produced the expected PMPE-specific ˜3.0 Kbpamplimer.

The ˜3.0 Kbp PMPE amplimer was purified from unincorporated primersusing hydroxyapatite spin columns (QiaGen) and digested to completionwith an excess of SalI (BRL, ˜10 units per 1 μg DNA) according to themanufacturers recommendations. The purified and digested PMPE ORF wasthen purified via QiaGen columns as described above and cloned intoplasmid pQE-30 that had been previously digested to completion with SalIand treated with calf intestinal alkaline phosphatase (BRL, ˜0.05units/pmole of 5′ ends) to prevent vector religation (˜5:1,insert:vector ratio).

Digestion of the pQE-30 vector with SalI produces a single restrictionfragment of ˜3.4 Kbp.

Aliquots from the ligation reaction were then used to electrotransform asuitable E. coli host (e.g., M15 (pREP)). Transformed cells were platedon 2X-YT agar containing 100 μg/ml ampicillin (for pQE-30) and 40 μg/mlkanamycin (for pREP) grown at 37° C. for ˜12-16 hours. Mini-prep DNAfrom ampicillin/kanamycin-resistant transformants picked at random wereprepared using commercially available reagents (QiaGen Mini Prep Kit)and examined for the presence of recombinant plasmids larger than thepQE-30 plasmid (i.e., insert-carrying plasmids). Putative PMPE-insertcarrying recombinant plasmids were then digested to completion with SalIand examined for the presence of the PMP-specific fragment by standardagarose gel electrophoresis (0.8% agarose, TAE buffer). All plasmidstested were found to contain the PMPE insert. Plasmid M15 pREP(pQE-pmpE-CT)#37 was one recombinant derivative isolated by theseprocedures.

6.12. PCR Cloning of the PMPI ORF

To produce high levels of recombinant PMPI protein for immunogenicityand protective efficacy studies, the PMPI ORF was PCR cloned into an E.coli high expression vector pBAD/Thio-E (Invitrogen) under the controlof the araBAD promoter. Oligonucleotide PCR primers complementary to theDNA sequences encoding the first 10 amino acid residues of theN-terminus and the last 8 C-terminal amino acid residues of the C.trachomatis L2 PMPI ORF present in the UC Berkeley genomic database (seeStephens et al., 1998, Science: 282: 754-759 and GenBank Accession No.NC000117) were synthesized. In addition to the PMPI specific sequences,the 5′ forward PCR primer contained the sequence 5′ AGG CAG AGG CAT-3′(SEQ ID NO.:68) immediately upstream of the PMPI ATG start codon whilethe 3′ reverse primer contained the sequence 5′-AGG CAG AGG GTC GAC′3′(SEQ ID NO.:70) immediately downstream of the C-terminal GAA codon. Theoligonucleotides (SEQ ID NO.:69 and 70) were used to amplifyPMPI-specific PCR products from C. trachomatis L2 genomic DNA. Thesequence of the amplification primers for SEQ ID NO.:69 (forward primer)and SEQ ID NO.:70 (reverse primer) used for these PCR reactions areshown below. Forward primer PMPI (SEQ ID NO.: 69) 5′- AGG CAG AGG CATATG CGA CCT GAT CAC ATG AAC TTC TGT TG- 3′ Reverse primer PMPI (SEQ IDNO.: 70) 5′-AGG CAG AGG GTC GAC GAA CCT GTA AGT GGT CCC CAG ATC -3′

Standard PCR amplification reactions (2 mM Mg²⁺, 200 μmol dNTPs, 0.75units AmpliTaq, 50 l final volume) were programmed using ˜0.1 μg of C.trachomatis L2 chromosomal DNA. Amplification of the PMPI targetsequence was achieved using a standard 32-cycle, three-step thermalprofile, i.e., 95° C., 30 sec.; 55° C., 45 sec.; 72° C., 3 min.Amplification was carried out in 0.2 ml polypropylene thin-walled PCRtubes (Perkin-Elmer) in a Perkin-Elmer model 2400 thermal cycler. PCRamplification reactions produced the expected PMPI-specific ˜3.0 Kbpamplimer.

The ˜3.0 Kbp PMPI amplimer was purified from unincorporated primersusing hydroxyapatite spin columns (QiaGen). The purified PMPI ORF wasthen cloned into the T/A PCR product cloning vector pUni/V5-HIS-TOPOplasmid (Invitrogen) using standard TOPO cloning methods described bythe manufacturer. The plasmid pUni/V5-His-TOPO carries a loxP siteupstream of the T/A cloning site that can be used to recombine insertcarrying plasmids with other loxP-containing plasmids for subsequentcontrolled expression of recombinant protein.

Aliquots from the PMPI amplimer and pUni/V5-His-TOPO ligation reactionwere then used to transform a suitable E. coli host (e.g., PIR1).Transformed cells were plated on 2X-YT agar containing 40 μg/mlkanamycin and grown at 37° C. for ˜12-16 hours. Mini-prep DNA fromkanamycin-resistant transformants picked at random were prepared usingcommercially available reagents (QiaGen Mini Prep Kit) and examined forthe presence of recombinant plasmids larger than the ˜2.2 Kbp vectorplasmid Uni/V5-His-TOPO (i.e., insert-carrying plasmids).pUni/V5-His-TOPO plasmids carrying the cloned PMPI ORF were mixed (˜100ng) with the plasmid pBAD/Thio-E (˜100 ng) in the presence of arecombination buffer supplied by the manufacturer and the two wererecombined into a single molecule by homologous recombination at the loxsites on both the pUni/V5-His-TOPO-PMPI plasmid and pBAD/Thio-E vectorcatalyzed by the Cre recombinase. pBAD/Thio-E carries a segment of theE. coli thioredoxin protein (˜12 kD) that has been mutated to encode anN-terminal (His) affinity chromatography domain. A single loxPrecombination site is also encoded by pBAD/Thio-E immediately downstreamof the His-Thio redoxin coding sequence. The His-Thioredoxin codingsequence and the loxP site are themselves downstream of the araBADpromoter. Recombining pBAD/Thio-E with PMPI derivatives ofpUni/V5-His-TOPO through the loxP sites using the loxP-specific Crerecombinase results in the formation of a chimeric protein with theHis-thioredoxin domain at the N-terminus which is fused in frame to thePMPI ORF. Following recombination the chimeric plasmids were transformedinto E. coli TOP10 cells. Transformed cells were selected on platescontaining 40 μg/ml kanamycin. All ˜9.5 kBp plasmids tested were foundto contain the PMPI insert. Plasmid TOP10 (pBAD-PmpI-Ct-Uni)#7 was onerecombinant derivative isolated by these procedures.

6.13. Expression of Recombinant PMPE

One milliliter of a frozen stock of E. coli strain M15 pREP containingplasmid (pQE-pmpE-CT)#37 was used to inoculate ˜100 ml of 2X-YT brothcontaining 40 μg/ml kanamycin and 100 μg/ml ampicillin and grownovernight at 30° C. to prepare a fermentor seed culture. Approximately20 ml of the overnight seed culture was then used to inoculate a NewBrunswick Bioflow 3000 fermentor loaded with ˜2.0 L of 2X-YT brothcontaining 40 μg/ml kanamycin and 100 μg/ml ampicillin. The culture wasgrown at 37° C. with vigorous aeration until an O.D.₆₀₀ value of −1.0was attained. Expression of rPMPE was induced by adding IPTG to a 1.0 mMfinal concentration and continuing fermentation. Incubation in thepresence of IPTG was continued for approximately 4-5 hours.

At the end of the induction period, the E. coli culture, with some cellsdisplaying classic recombinant protein inclusion bodies, was harvestedby continuous flow centrifugation using an Heraeus Contifuge 28RScentrifuge. Following centrifugation, cell mass was scraped from thecentrifuge bowl and stored at −70° C. until processed.

Approximately 15 gm of the M15 pREP (pQE-pmpE-CT)#37 frozen cell pastewas resuspended by vortexing and trituration in ˜40 ml of ice cold 10 mMsodium phosphate buffer, pH 7.3. Once suspended, lysozyme (Chicken eggwhite, Sigma) and DNase I (Bovine pancreas, Sigma) were added to finalconcentrations of 1.0 mg/ml and 0.01 mg/ml, respectively, and themixture incubated on ice for 30-45 minutes. Cells were disrupted by 2sequential passes through a pre-cooled (˜4° C.) SLM Aminco FrenchPressure Cell (˜14 Kpsi, 1″-diameter bore). The cell lysate was thencentrifuged for 5 min at ˜500×g (4° C.) in a Sorvall SS34 rotor toremove unbroken cells. Insoluble material containing the rPMPE wasisolated (pelleted) by centrifugation for 45 minutes at ˜20,000×g (4°C.) in a Sorvall SS34 rotor. The supernatant from this centrifugationwas discarded and the insoluble fraction stored at −20° C. in pelletform.

To selectively extract contaminating proteins and remove endotoxin, therPMPE-containing insoluble pellet was thawed on ice and washed twicewith 10 ml of PBS buffer containing 2.0% Triton X-100. Washing wasperformed at room temperature and suspension of the gelatinousrPMPE-containing pellet was accomplished by vortexing and homogenizationin a conventional glass tissue grinder. Insoluble material containingthe rPMPE was recovered after washing by centrifugation at ˜10,000×g for20 minutes (room temperature) in a Sorvall SS34 rotor. Insolublematerial was then washed (again by vortexing and homogenization) 2 timeswith 10 ml of a 4.0 M urea solution containing 2.0 M NaCl. Washed rPMPEmaterial was recovered by centrifugation as above. The insoluble rPMPEfraction was further washed 2 times with 10 ml of a PBS solutioncontaining 1.0% Zwittergent 3-14 (Sigma).

The rPMPE pellet recovered after centrifugation of the final washsolution was then solubilized for 2 hours at room temperature instandard Laemelli SDS-PAGE sample buffer containing 4 M urea.Solubilized rPMPE was size fractionated into a single protein band of˜105 kDa by electrophoresis through a cylinderical ˜37 mm×˜12 cm 6%polyacrylamide (36:1, acrylamide:bis-acrylamide) Tris/glycine/SDSpreparative gel. A 4% polyacrylamide stacking gel was formed on top ofthe resolving gel (˜37 mm×˜3 cm). Electrophoresis was carried out on aBioRad model 491 PrepCell unit for ˜12 hours at ˜22° C. (12 wattsconstant wattage) using a conventional Tris/glycine/SDS running buffer(BioRad). As electrophoresis proceeded, size fractionated proteinseluting from the bottom of the PrepCell were pumped to an Amershamfraction collector where ˜8.0 ml fractions were collected.

Aliquots (˜20 μl) from each fraction were mixed with an equal volume of2× LSB heated to ˜100° C. for 3 minutes and electrophoresed on a 4-20%SDS-PAG to identify fractions containing the PMPE protein. Prestainedmolecular weight standards were loaded into a parallel lane and wereused as a size gauge. PMPE-containing factors were pooled and excess SDSdetergent was removed by diluting the eluted sample with approximately20 volumes of sterile, endotoxin-free 10 mM sodium phosphate buffer (pH7.4) and concentrated to approximately 1.0 mg/ml by ultrafiltration inan Amicon stirred concentration cell using a YM30 ultrafiltrationmembrane.

Residual endotoxin was removed from the concentrated rPMPE solution bypolymyxin B Affi-Prep Polymyxin Matrix (BioRad) treatment. Affi-Preptreatment was performed overnight at 4° C. in a batch mode according tothe manufacturers recommendations.

The protein concentration of the concentrated, polymyxin B-treated rPMPEwas determined using the Micro BCA method (Pierce Chem.) and BSA as astandard.

Purified rPMPE (˜0.9-1.2 mg/ml protein concentration) was evaluated forpurity, identity, and residual endotoxin burden by SDS-PAGE, Westernblot, and a colorimetric endotoxin assay (BioWhittaker), respectively.The gel-purified rPMPE material displayed a purity of >95% as a singleband of the expected molecular size by gel analysis. Residual endotoxinis calculated to be ≦0.05 EU/g.

6.14. Expression of Recombinant PMPI

One milliliter of a frozen stock of E. coli strain TOP10 containingplasmid (pBAD-pmpI-Ct-Uni)#7 was used to inoculate ˜100 ml of 2X-YTbroth containing 40 μg/ml kanamycin and grown overnight at 37° C. toprepare a fermenter seed culture. Approximately 20 ml of the overnightseed culture was then used to inoculate a New Brunswick Bioflow 3000fermenter loaded with ˜2.0 L of 2X-YT broth containing 40 μg/mlkanamycin. The culture was grown at 37° C. with vigorous aeration untilan O.D.₆₀₀ value of C1.0 was attained. Expression of rPMPI was inducedby addition of arabinose to a final concentration of 1.0 mM andcontinuing cultivation for approximately 4-5 hours.

At the end of the induction period, the E. coli culture, with some cellsdisplaying classic recombinant protein inclusion bodies, was harvestedby continuous flow centrifugation using an Heraeus Contifuge 28RScentrifuge. Following centrifugation, cell mass was scraped from thecentrifuge bowl and stored at −70° C. until processed.

Approximately 15 gm of the TOP10 (pBAD-pmpI-Ct-Uni)#7 frozen cell pastewas resuspended by vortexing and trituration in ˜40 ml of ice cold 10 mMsodium phosphate buffer, pH 7.3. Once suspended, lysozyme (Chicken eggwhite, Sigma) and DNAase I (Bovine pancreas, Sigma) were added to finalconcentrations of 1.0 mg/ml and 0.01 mg/ml, respectively, and themixture incubated on ice for 30-45 minutes. Cells were disrupted by 2sequential passes through a pre-cooled (˜4° C.) SLM Aminco FrenchPressure Cell (˜14 Kpsi, 1″ diameter bore). The cell lysate was thencentrifuged for 5 min at ˜500×g (4° C.) in a Sorvall SS34 rotor toremove unbroken cells. Insoluble material containing the rPMPI wasisolated (pelleted) by centrifugation for 45 min at ˜20,000×g (4° C.) ina Sorvall SS34 rotor. The supernatant from this centrifugation wasdiscarded and the insoluble fraction stored at −20° C. in pellet form.

To selectively extract contaminating proteins and remove endotoxin, therPMPI-containing insoluble pellet was thawed on ice and washed twicewith 10 ml of PBS buffer containing 2.0% Triton X-100. Washing wasperformed at room temperature and suspension of the gelatinousrPMPI-containing pellet was accomplished by vortexing and homogenizationin a conventional glass tissue grinder. Insoluble material containingthe rPMPI was recovered after washing by centrifugation at ˜10,000×g for20 minutes (room temperature) in a Sorvall SS34 rotor. Insolublematerial was then washed (again by vortexing and homogenization) 2 timeswith 10 ml of a 4.0 M urea solution containing 2.0 M NaCl. Washed rPMPImaterial was recovered by centrifugation as above. The insoluble rPMPIfraction was further washed 2 times with 10 ml of a PBS solutioncontaining 1.0% Zwittergent 3-14 (Sigma).

The rPMPI pellet recovered after centrifugation of the final washsolution was then solubilized for 2 hours at room temperature instandard Laemelli SDS-PAGE sample buffer containing 4 M urea.Solubilized rPMPI-thiroredoxin-fusion protein was size fractionated intoa single protein band of ˜105 kDa using a model 491 PrepCell (BioRad)cylindrical gel as described above for PMPE above. PMPI was obtainedfrom SDS PAG and residual endotoxin removed as described for PMPE above.

The protein concentration of the concentrated, polymyxin B-treatedrPMPI-thioredoxin fusion protein was determined using the Micro BCAmethod (Pierce Chem.) and BSA as a standard.

Purified rPMPI (˜0.9-1.2 mg/ml protein concentration) was evaluated forpurity, identity, and residual endotoxin burden by SDS-PAGE, Westernblot, and a colorimetric endotoxin assay (BioWhittaker), respectively.The gel-purified rPMPI material displayed a purity of >95% as a singleband of the expected molecular size by gel analysis. Residual endotoxinis calculated to be ≦0.05 EU/g

6.15. Affinity Chromatography Purification of Recombinant Protein

Recombinant PMPE or PMPI protein is purified to apparent homogeneityusing standard preparative immobilized metal affinity chromatography(MAC) procedures. Briefly, an E. coli strain harboring an expressionplasmid containing PMPE or PMPI protein gene is grown in Luria broth ina 5 L fermenter (New Brunswick) at 37° C. with moderate aeration untilmid-log phase (˜0.5 O.D.₆₀₀) and induced with IPTG or arabinose (1 mMfinal) for 4-5 hours. Cell paste is collected, washed in PBS and storedat −20° C. Aliquots of frozen cell paste (˜9-10 grams wet weight) aresuspended in ˜120 ml of D-PBS by mechanical agitation and lysed bypassage through a French pressure cell (2×, 14,000 psi, 4° C.). Solubleprotein is then removed from rPMPE or rPMPI protein inclusion bodies byhigh speed centrifugation (˜20,000×g, 4° C., 30 min).

The insoluble pellet containing rPMPE or rPMPI protein is suspended in˜20 ml of ice cold D-PBS by homogenization and centrifuged as above.Washed rPMPE or rPMPI protein inclusion bodies are then denatured bysuspension in a sodium phosphate buffer (0.1 M, pH 7.0) containing 7.4 Mguanidine hydrochloride, 5% 2-ME and 10% glycerol and loaded onto aNi²⁺-affinity column (1.5 cm×25 cm, bed volume ˜30 ml) prepared fromFast-Flow Chelating Sepharose (Pharmacia) and charged with Ni²⁺ or Zn²⁺ions by standard procedures. Unbound material is removed by washing thecolumn with ˜5-10 column volumes of a sodium phosphate buffer (0.1 M, pH7.0) containing ˜7.4 M guanidine HCl.

Recombinant PMPE or PMPI protein bound to the affinity resin by virtueof the (His)₆ affinity purification domain is eluted using sodiumphosphate buffer (pH 7.4) containing 7.4 M guanidine HCl and 200 mMimidazole. Eluted material is dialyzed against TE buffer containing SDS(0.5%) to remove the guanidine. Dialyzed material is concentrated usinga Amicon stirred cell concentrator using a YM30 membrane and mixed witha ⅕ volume of 5×SDS gel sample buffer containing 1 mM 2-mercaptoethanoland boiled at 100° C. for 5 minutes.

Samples are loaded onto Tris/glycine acrylamide gels (4% stacking gel,4-20% resolving gel, 30:0.8 acrylamide:bis solution, 1 mm thickness). Aprestained molecular weight standard (See Blue or Multimark, Novex) isrun in parallel with the rPMPE or rPMPI protein samples to identify sizefractions on the gel. The purity of rPMPE or rPMPI protein is determinedusing conventional SDS-PAGE and commercially available silver stainingreagents (Silver Stain Plus, Novex).

6.16. Generation of a Radiolabeled Screening Probe

The sequence information shown above is used to design a pair ofnondegenerate oligonucleotide primers. PCR amplification of DNAfragments is performed under the same conditions as described above withthe exception that the annealing temperature is lowered to 50° C. TheDNA fragment is isolated from an agarose gel as before and radiolabeledusing [³²P]-gamma-ATP and T4 polynucleotide kinase according to standardmethods. Unincorporated radiolabel is separated from the probe on a G25Sepharose spin column. Before use, the probe is denatured for 2 min. at95° C. and subsequently chilled on ice (4° C.).

6.17. Hybridization of Plaque-Lift Filters and Southern Blots withRadiolabeled Probe

Phage plaques from library platings are immobilized on nylon filtersusing standard transfer protocols well known to those skilled in theart. Digested bacterial genomic DNA, phage or plasmid DNA iselectrophoresed on 0.8% TAE-agarose gels and transferred onto nylonfilters using a pressure blotter (Stratagene) according to themanufacturer's recommendations. Hybridizations with selected probes areperformed at 37° C. (for example, to detect PMP homologs of otherspecies). Hybridizations with specific probes are generally carried outat 50-60° C. (e.g., to detect the identical sequence or the analogousprotein in another serovar). Washes of increasing stringency are done atthe respective hybridization temperatures until nonspecific backgroundis minimized.

6.18. Construction of a Chlamydia Genomic DNA Library

A genomic library was constructed in the λZAPII replacement vectorobtained from Stratagene. The vector arms were digested with EcoR1.Digests of Chlamydia DNA by EcoR1 were performed to yield fragment sizesbetween 1 kb and 5 kb. Ligations of vector arms and insert DNA werecarried out according to standard protocols. Ligation reactions werepackaged in vitro using the Stratagene GigaPack Gold III extract. Thepackaged phage were plated on E. coli X1 Blue MRA (P2) (Stratagene). Aninitial library titer was determined and expressed as number of pfu.

The library is screened using 4×10⁴ pfu that are plated at a density of8×10³ pfu/130 mm plate with a PMPE or PMPI specific, probe. Severalputative positive phage plaques are located and the strongesthybridizing phage are eluted from cored agarose plugs, titered andreplated for secondary screening. The selected phage are replated at lowdensity (approximately 100 pfu/plate) and plaques are analyzed by PCRusing primer pairs. Inserts carrying plasmids (phagemids) are rescuedfrom the selected phage by co-infecting E. coli cells with anappropriate helper virus.

6.19. Determination of Insert Size and Mapping of DNA Fragments

In order to estimate the size of inserts, phagemid DNA is digested withappropriate restriction enzymes (e.g., NotI) and the digests areanalyzed on a 0.5% TAE-agarose gel side by side with suitable DNAmarkers. In order to map restriction fragments that would hybridize tothe probe, DNA from phagemid isolates is digested with a number ofcommon restriction enzymes either alone or in combination with NotI. Therationale of this approach is to discriminate between fragments thatspan the insert/phagemid vector junction and those that map on the NotIinsert. The series of single and double digests are run side-by-side foreach phage isolate and analyzed by Southern analysis with radiolabeledprobe.

6.20. Sequencing of the PMPE and PMPI Gene

Sequencing of the PMPE and PMPI gene is performed using the DyeTerminator Cycle Sequencing Kit from Perkin-Elmer according to themanufacturer's specifications. The sequencing reactions are read usingan ABI Prism 310 Genetic Analyzer. The sequences are aligned using theAutoAssembler software (Perkin-Elmer) provided with the ABI Prism 310sequencer.

The sequence of the nucleic acid encoding the PMPE protein and thededuced amino acid sequence of the PMPE expressed by Plasmid M15 pREP(pQE-pmpE-Ct-Uni)#37 are shown in FIG. 8 (SEQ ID Nos.:72 and 73).

6.21. In Vivo Efficacy of PMPE

The ability of PMPE to protect female C3HeOUJ mice using the proceduredisclosed in Section 6.9.2, supra, is shown in Table 4. Groups of micewere immunized intranasally (i.n.) with 10 μg PMPE (with or without ˜5μg mLT as an adjuvant) prior to challenge with live C. trachomatisserovar F. Negative control mice were immunized with ˜5 μg mLTintranasally prior to administration of live C. trachomatis. Positivecontrol groups were immunized with adjuvant alone intranasally but werenot administered live C. trachomatis. The fertility rate for micevaccinated with PMPE or PMPE and mLT was 50% and 46% respectively. Thefertility rate for negative control mice inunized with adjuvant alone(mLT) was 9% and the fertility of positive control mice not infectedwith C. trachomatis but administered mLT was 95%. These resultsdemonstrate that PMPE is an effective vaccine for amelioratinginfertility induced by infection with C. trachomatis. Thus, one withskill would in light of teaching of the specification be able to makethe claimed PMPE polypeptides and determine if the PMPE polypeptideshave the ability to ameliorate disease associated with infection with C.trachomatis. TABLE 4 FERTILITY ASSESSMENT FOR PMPE Group Vaccine andRoute % Fertility I PMPE + mLT/i.n. 50 II PMPE/i.n. 46 III mLT/i.i.(Neg. 9 control) IV mLT/i.n. (Poss. 95 control)

The present invention is not to be limited in scope by the microorganismdeposited or the specific embodiments described herein. It will beunderstood that variations which are functionally equivalent are withinthe scope of this invention. Indeed, various modifications of theinvention, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-57. (canceled)
 58. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: (a) amino acids 32-965of SEQ ID NO: 2; (b) SEQ ID NO: 2; (c) amino acids 125-190 of SEQ ID NO:2; (d) amino acids 590-625 of SEQ ID NO: 2; (e) amino acids 1-500 of SEQID NO: 2; (f) an amino acid sequence encoded by the Chlamydia insert inE.coli M15 assigned as ATCC PTA No. 2462; and (g) a combination of twoor more amino acid sequences of (a)-(f).
 59. The polypeptide of claim58, further comprising a heterologous polypeptide.
 60. The polypeptideof claim 59, wherein said heterologous polypeptide is selected from thegroup consisting of a pre- and/or pro-sequence, an affinity purificationsequence, and an immunogenic sequence.
 61. The polypeptide of claim 59,wherein said heterologous polypeptide is selected from the groupconsisting of SEQ ID NO: 4, amino acids 13-40 of SEQ ID NO: 4; aminoacids 70-110 of SEQ ID NO: 4; amino acids 150-225 of SEQ ID NO: 4; aminoacids 250-290 of SEQ ID NO: 4; amino acids 370-455 of SEQ ID NO: 4;amino acids 400-455 of SEQ ID NO: 4; amino acids 470-520 of SEQ ID NO:4; amino acids 615-670 of SEQ ID NO: 4; amino acids 710-775 of SEQ IDNO: 4; amino acids 765-825 of SEQ ID NO: 4; amino acids 830-860 of SEQID NO: 4; amino acids 1-500 of SEQ ID NO: 34; and an amino acid sequenceencoded by a Chlamydia insert in E.coli TOP10 assigned as ATCC PTA No.2461.
 62. An isolated antibody or an antigen-binding fragment thereofthat specifically binds the polypeptide of claim
 58. 63. An isolatedpolynucleotide comprising a nucleic acid sequence, which encodes thepolypeptide of claim
 58. 64. The polynucleotide of claim 63, whereinsaid nucleic acid sequence is selected from the group consisting of: (a)nucleotides 94-2895 of SEQ ID NO: 1; (b) SEQ ID NO:1, (c) a Chlamydiainsert in E.coli M15 assigned as ATCC PTA No. 2462; and (d) acombination of (a), (b), or (c), or the complement thereof.
 65. A vectorcomprising the polynucleotide of claim
 63. 66. A host cell comprisingthe vector of claim
 65. 67. A composition comprising the polypeptide ofclaim 58 and a carrier.
 68. A composition comprising the antibody orantigen-binding fragment thereof of claim 62 and a carrier.
 69. Acomposition comprising the polynucleotide of claim 63 and a carrier. 70.The composition of claim 67, further comprising an adjuvant.
 71. Thecomposition of claim 67, further comprising an immunogen selected fromthe group consisting of a lipid, a lipoprotein, a phospholipid, alipooligosaccharide, a protein, an attenuated or inactivated organism,and a combination of two or more of said immunogens.
 72. A method ofproducing an immune response in an animal comprising administering to ananimal in need thereof the composition of claim
 67. 73. A method ofpreventing, treating or ameliorating a disorder or disease associatedwith a Chlamydia infection comprising administering to a subject in needthereof an effective amount of the composition of claim
 67. 74. Acomposition comprising an antagonist which inhibits an activity of thepolypeptide of claim
 58. 75. A method for identifying a compound thatinteracts with the polypeptide of claim 58, wherein said methodcomprising contacting the polypeptide of claim 58 with a compound to bescreened under conditions that permit interaction between said compoundand said polypeptide; and detecting the interaction of said compoundwith said polypeptide.
 76. A method for identifying a compound whichinteracts with the polynucleotide of claim 63, wherein said methodcomprising contacting the polynucleotide of claim 63 or a complementthereof with a compound to be screened under conditions that permitinteraction between said compound and said polynucleotide; and detectingthe interaction of said polynucleotide with said compound.
 77. Anisolated polypeptide comprising an amino acid sequence of SEQ ID NO: 4or an amino acid sequence encoded by a Chlamydia insert in E. coli TOP10assigned as ATCC PTA No.
 2461. 78. The polypeptide of claim 77, furthercomprising a heterologous polypeptide.
 79. The polypeptide of claim 78,wherein said heterologous polypeptide is selected from the groupconsisting of a pre- and/or pro-sequence, an affinity purificationsequence, and an immunogenic sequence.
 80. The polypeptide of claim 78,wherein said heterologous polypeptide is selected from the groupconsisting of SEQ ID NO: 2, amino acids 15-56 of SEQ ID NO: 2; aminoacids 15-121 of SEQ ID NO: 2; amino acids 45-125 of SEQ ID NO: 2; aminoacids 125-190 of SEQ ID NO: 2; amino acids 195-261 of SEQ ID NO: 2;amino acids 275-366 of SEQ ID NO: 2; amino acids 375-440 of SEQ ID NO:2; amino acids 440-490 of SEQ ID NO: 2; amino acids 525-590 of SEQ IDNO: 2; amino acids 590-625 of SEQ ID NO: 2; amino acids 615-650 of SEQID NO: 2; amino acids 625-700 of SEQ ID NO: 2; amino acids 725-800 ofSEQ ID NO: 2; amino acids 755-775 of SEQ ID NO: 2; amino acids 785-845of SEQ ID NO: 2; amino acids 815-865 of SEQ ID NO: 2; amino acids 1-31of SEQ ID NO: 2; amino acids 1-500 of SEQ ID NO: 2; and an amino acidsequence encoded by a Chlamydia insert in E. coli M15 assigned as ATCCPTA No.
 2462. 81. An isolated antibody or an antigen-binding fragmentthereof that specifically binds the polypeptide of claim
 77. 82. Anisolated polynucleotide comprising a nucleic acid sequence, whichencodes the polypeptide of claim
 77. 83. The polynucleotide of claim 82,wherein said nucleic acid sequence is SEQ ID NO:3 or the Chlamydiainsert in E. coli TOP10 assigned as ATCC PTA No.
 2461. 84. A vectorcomprising the polynucleotide of claim
 82. 85. A host cell comprisingthe vector of claim
 84. 86. A composition comprising the polypeptide ofclaim 77 and a carrier.
 87. A composition comprising the polynucleotideof claim 82 and a carrier.
 88. A composition comprising the antibody orantigen-binding fragment thereof of claim 81 and a carrier.
 89. Thecomposition of claim 86, further comprising an adjuvant.
 90. Thecomposition of claim 86, further comprising an immunogen selected fromthe group consisting of a lipid, a lipoprotein, a phospholipid, alipooligosaccharide, a protein, an attenuated or inactivated organism,and a combination of one or more of said immunogens.
 91. A method ofproducing an immune response in an animal comprising administering to ananimal in need thereof the composition of claim
 86. 92. A method ofpreventing, treating or ameliorating a disorder or disease associatedwith a Chlamydia infection comprising administering to a subject in needthereof an effective amount of the composition of claim
 86. 93. Acomposition comprising an antagonist which inhibits an activity of thepolypeptide of claim
 77. 94. A method for identifying a compound thatinteracts with the polypeptide of claim 77, wherein said methodcomprising contacting the polypeptide of claim 77 with a compound to bescreened under conditions that permit interaction between said compoundand said polypeptide; and detecting the interaction of said compoundwith said polypeptide.
 95. A method for identifying a compound whichinteracts with the polynucleotide of claim 82, wherein said methodcomprising contacting the polynucleotide of claim 82 with a compound tobe screened under conditions that permit interaction between saidcompound and said polynucleotide; and detecting the interaction of saidpolynucleotide with said compound.
 96. An isolated polypeptideconsisting essentially of an amino acid sequence at least 95% identicalto a reference amino acid sequence selected from the group consistingof: amino acids 15-56 of SEQ ID NO: 2; amino acids 15-121 of SEQ ID NO:2; amino acids 45-125 of SEQ ID NO: 2; amino acids 125-190 of SEQ ID NO:2; amino acids 195-261 of SEQ ID NO: 2; amino acids 275-366 of SEQ IDNO: 2; amino acids 375-440 of SEQ ID NO: 2; amino acids 440-490 of SEQID NO: 2; amino acids 525-590 of SEQ ID NO: 2; amino acids 590-625 ofSEQ ID NO: 2; amino acids 615-650 of SEQ ID NO: 2; amino acids 625-700of SEQ ID NO: 2; amino acids 725-800 of SEQ ID NO: 2; amino acids755-775 of SEQ ID NO: 2; amino acids 785-845 of SEQ ID NO: 2; aminoacids 815-865 of SEQ ID NO: 2; amino acids 1-31 of SEQ ID NO: 2; andamino acids 1-500 of SEQ ID NO: 2, wherein said polypeptide specificallybinds an antibody raised against a polypeptide consisting of SEQ ID NO:2.
 97. The polypeptide of claim 96, wherein said amino acid sequence isidentical to said reference amino acid sequence.
 98. The polypeptide ofclaim 96, further comprising a heterologous polypeptide.
 99. Thepolypeptide of claim 98, wherein said heterologous polypeptide isselected from the group consisting of a pre- and/or pro-sequence, anaffinity purification sequence, and an immunogenic sequence.
 100. Thepolypeptide of claim 98, wherein said heterologous polypeptide isselected from the group consisting of SEQ ID NO: 4, amino acids 13-40 ofSEQ ID NO: 4; amino acids 70-110 of SEQ ID NO: 4; amino acids 150-225 ofSEQ ID NO: 4; amino acids 250-290 of SEQ ID NO: 4; amino acids 370-455of SEQ ID NO: 4; amino acids 400-455 of SEQ ID NO: 4; amino acids470-520 of SEQ ID NO: 4; amino acids 615-670 of SEQ ID NO: 4; aminoacids 710-775 of SEQ ID NO: 4; amino acids 765-825 of SEQ ID NO: 4;amino acids 830-860 of SEQ ID NO: 4; amino acids 1-500 of SEQ ID NO: 34;and an amino acid sequence encoded by a Chlamydia insert in E. coliTOP10 assigned as ATCC PTA No.
 2461. 101. An isolated polynucleotidecomprising a nucleotide sequence, which encodes the polypeptide of claim96.
 102. A vector comprising the polynucleotide of claim
 101. 103. Ahost cell comprising the vector of claim
 102. 104. A compositioncomprising the polypeptide of claim 96 and a carrier.
 105. A compositioncomprising the polynucleotide of claim 101 and a carrier.
 106. Thecomposition of claim 104, further comprising an adjuvant.
 107. Thecomposition of claim 104, further comprising an immunogen selected fromthe group consisting of a lipid, a lipoprotein, a phospholipid, alipooligosaccharide, a protein, an attenuated or inactivated organism,and a combination of two or more of said immunogens.
 108. A method ofproducing an immune response in an animal comprising administering to ananimal in need thereof the composition of claim
 104. 109. A method ofpreventing, treating or ameliorating a disorder or disease associatedwith a Chlamydia infection comprising administering to a subject in needthereof an effective amount of the composition of claim
 104. 110. Amethod for identifying a compound that interacts with the polypeptide ofclaim 96, wherein said method comprising contacting the polypeptide ofclaim 96 with a compound to be screened under conditions that permitinteraction between said compound and said polypeptide; and detectingthe interaction of said compound with said polypeptide.
 111. A methodfor identifying a compound which interact with the polynucleotide ofclaim 101, said method comprising contacting the polynucleotide of claim101 with a compound to be screened under conditions that permitinteraction between the compound and the polynucleotide; and detectingthe interaction of the polynucleotide with the compound.
 112. Anisolated polypeptide consisting essentially of an amino acid sequence atleast 95% identical to a reference amino acid sequence selected from thegroup consisting of: amino acids 13-40 of SEQ ID NO: 4; amino acids70-110 of SEQ ID NO: 4; amino acids 150-225 of SEQ ID NO: 4; amino acids250-290 of SEQ ID NO: 4; amino acids 370-455 of SEQ ID NO: 4; aminoacids 400-455 of SEQ ID NO: 4; amino acids 470-520 of SEQ ID NO: 4;amino acids 615-670 of SEQ ID NO: 4; amino acids 710-775 of SEQ ID NO:4; amino acids 765-825 of SEQ ID NO: 4; amino acids 830-860 of SEQ IDNO: 4; and amino acids 1-500 of SEQ ID NO: 34, wherein said polypeptidespecifically binds to antibodies raised against a polypeptide consistingof SEQ ID NO:
 4. 113. The polypeptide of claim 112, wherein said aminoacid sequence is identical to the reference amino acid sequence. 114.The polypeptide of claim 112, further comprising a heterologouspolypeptide.
 115. The polypeptide of claim 114, wherein saidheterologous polypeptide is selected from the group consisting of a pre-and/or pro-sequence, an affinity purification sequence, and animmunogenic sequence.
 116. The polypeptide of claim 114, wherein saidheterologous polypeptide is selected from the group consisting of SEQ IDNO: 2, amino acids 15-56 of SEQ ID NO: 2; amino acids 15-121 of SEQ IDNO: 2; amino acids 45-125 of SEQ ID NO: 2; amino acids 125-190 of SEQ IDNO: 2; amino acids 195-261 of SEQ ID NO: 2; amino acids 275-366 of SEQID NO: 2; amino acids 375-440 of SEQ ID NO: 2; amino acids 440-490 ofSEQ ID NO: 2; amino acids 525-590 of SEQ ID NO: 2; amino acids 590-625of SEQ ID NO: 2; amino acids 615-650 of SEQ ID NO: 2; amino acids625-700 of SEQ ID NO: 2; amino acids 725-800 of SEQ ID NO: 2; aminoacids 755-775 of SEQ ID NO: 2; amino acids 785-845 of SEQ ID NO: 2;amino acids 815-865 of SEQ ID NO: 2; amino acids 1-31 of SEQ ID NO: 2;amino acids 1-500 of SEQ ID NO: 2; and an amino acid sequence encoded bya Chlamydia insert in E.coli M15 assigned as ATCC PTA No.
 2462. 117. Anisolated polynucleotide comprising a nucleic acid sequence, whichencodes the polypeptide of claim
 112. 118. A vector comprising thepolynucleotide of claim
 117. 119. A host cell comprising the vector ofclaim
 118. 120. A composition comprising the polypeptide of claim 112and a carrier.
 121. A composition comprising the polynucleotide of claim117 and a carrier.
 122. The composition of claim 120, further comprisingan adjuvant.
 123. The composition of claim 120, further comprising animmunogen selected from the group consisting of a lipid, a lipoprotein,a phospholipid, a lipooligosaccharide, a protein, an attenuated orinactivated organism, and a combination of two or more of saidimmunogens.
 124. A method of producing an immune response in an animalcomprising administering to an animal in need thereof the composition ofclaim
 120. 125. A method of preventing, treating or ameliorating adisorder or disease associated with a Chlamydia infection comprisingadministering to a subject in need thereof an effective amount of thecomposition of claim
 120. 126. A compound comprising an antagonist whichinhibits an activity of the polypeptide of claim
 112. 127. A method foridentifying a compound that interacts with the polypeptide of claim 112,wherein said method comprising contacting the polypeptide of claim 112with a compound to be screened under conditions that permit interactionbetween said compound and said polypeptide; and detecting theinteraction of said compound with said polypeptide.
 128. A method foridentifying a compound which interact with the polynucleotide of claim117, said method comprising contacting the polynucleotide of claim 117with a compound to be screened under conditions that permit interactionbetween the compound and the polynucleotide; and detecting theinteraction of the polynucleotide with the compound.